Angela A. Garibaldi Week 7

=Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human anti-body=

Introduction

 * HIV destroys CD4+ lymphocytes

Basic Structure

 * Viral envelope (responsible for entry of HIV to host cells)has glycoproteins which contain oligomeric spikes, trimeric spikes on the surface
 * gp41 (transmembrane envelope glycoprotein) anchors the spikes in the viral membrane
 * spike surface made of gp120 (exterior envelope glycoprotein) which have non-covalent interactions with each subunit of trimeric gp41 complex
 * 5 variable regions
 * 1-4 form loops exposed to the surface which are bonded at their bases by disulfide bonds.

Basic Entry and Binding

 * conserved regions of gp120 form discontinuous structures that are essential for interacting with the gp41 ectodomain, and target cell's viral receptors
 * gp120 glycoprotein is target for neutralizing antibodies elicited during normal infection
 * variable and conserved regions of gp120 highly glycosylated.
 * glycosylation and variability of gp120 important in antigen elicitation of an adaptive immune response and ability of a chemical structure "Antigen" to bind specifically with T cell receptors or Antibodies/B cell receptors of gp120 glycoprotein


 * immunodeficiency viruses enter primate host cells gp120 envelope glycoprotein binding to the CD4 glycoprotein (1ary receptor)
 * gp120 binds most amino-terminal domain of CD4 (1 of 4).


 * Important items for binding:
 * 1) CD4 structure similar/same to CDR2 region of immunoglobulins for gp120 binding
 * 2) conserved gp120 residues for CD4 binding


 * When CD4 binds it causes conformational changes in gp120, which expose or form binding sites for CCR5,CXCR4, which act as secondary receptors for viral entry.
 * exposures conserved gp120 structures also, detected by improved binding of gp120 antibodies which block binding of gp120-CD4 complexes to the chemokine receptor (referred to as CD4i, CD4-induced antibodies
 * CD4 causes conformational changes in envelope glycoproteins causing shedding of gp120 from complex


 * HIV and related retroviruses:
 * 1) require post-translational cleavage for activation
 * 2) transmembrane coat proteins (gp41 equiv) share similar sequences in N-terminal fusion peptides
 * 3) share common features of virus entry
 * 4) distinctive in modes of entry
 * 5) individualized receptors
 * 6) specialized exterior coat proteins (gp120 equiv)
 * 7) distinct mech. for receptor-mediated triggering of fusion

Structure determination

 * Crystallization by modifying protein surface
 * used gp120 from multiple different strains
 * Major steps utilized:
 * 1) deglosylation
 * 2) produce complexes using different ligands that would bind things together in different combinations
 * 3) screened ~20 variants/ligands to get crystals (ternary, truncated, N-terminal (2) domains (DID2) of CD4, Fab from human 17b.
 * 4) crystallized gp120 that was ultimately chosen from HXBc2 strain of HIV-1
 * 5) ternary structure solved by molecular replacement, isomorphous replacement, density modification

Structure of gp120

 * prolate ellipsoid 50x50x25 A, heart shaped
 * outer stacked double barrel that share hydrophobic core
 * (distal end/antiparallel 4 stranded) bridging sheet - excised V3, excursion via loop LF into β-hairpin, β20-β21 (H-bond with V1/V2 stem coming out of the inner domain)
 * This overall structure has nothing previous that it is similar too in structure.'
 * 1) no similarities when looking at inner domains
 * 2) missing terminal sections might prove this wrong in the future
 * 3) some similarities in outer domains to part of the promoter of FabA dehydrase, and dUTP pyrophosphatase (barrels).
 * 4) use as prototype due to considerable conservation rates (35%-51% depending on strain).
 * 5) Inner domain (of hydrophobic cores) more conserved than outer domain (Barrels)

Figure 1

 * side chain of Phe43 on CD4
 * N and C termini of gp120 are missing
 * gp41, viral membrane, located toward top of diagram
 * target membrane at base of diagram

Figure 2

 * 90 degree rotation of Fig 1 has viral membrane above, target membrane below, C-terminal tail of CD4 coming out of page
 * A and C' - spatial proximity between strands= main-chain H bonding
 * sequences with high variability in the loops are circled


 * B(topology diagram) represents orientation of a and c
 * C-stereo plot of an α-carbon trace
 * D is the structure based sequence alignment
 * 'Solvent accessibility noted by open/half-filled/filled circle system
 * Sequence variability noted by hash mark system
 * gp120 residues in "direct contact" with CD4 noted by an *, this way is stricter than the solvent accessible surface system

CD4-gp120 interaction

 * CD4 bound into a depression at the interface of outer/inner domain and bridging sheet of gp120, burying 742A2 of CD4 and 802A2 of gp120.
 * Only a few smaller surface areas touch.
 * Residues that touch span between 25-64 of CD4, distributed over 6 segments of gp120.
 * Residues that are important for binding do interact with each other. (Phe43,Arg59 of CD4)
 * Gp120 highly variable residues in contact with large water filled cavity that serves as a buffer zone (interfacial cavity)
 * gp120 residues in contact with CD4 only through main-chain atoms

Figure 3

 * A. Shows that Residue Phe 43 of CD4 reaches into heart of gp120 and Gp120 has recessed binding pocket
 * B. Phe43 of CD4 reaches up to contact cavity. Upper middle region has central unidentified density. Hydrophobic residues line back of cavity, around unidentified density.
 * C. Electrostatic potential at solvent-accessible surface
 * D. gp120 surface is within 3.5A surface to atom distance of CD4 (shown in yellow). This serves as imprint of CD4 on gp120 surface.
 * E. shows residues that are important for binding (Asp368,Glu370,Trp427,Asp457). Also shows the water filled cavity at the gp120-CD4 interface
 * F. Higher surface concentration of main-chain atoms in regions corresponding to CD4 imprint/contact
 * G. Scale of white (conserved) to red (highly variable)
 * H. Green shows secondary structure predictions that were incorrect
 * I. Shows 6 segments of gp120 (single lines) interacting with CD4 (double lines)
 * J. Hydrophobic interactions between Phe43 (CD4) and Trp 427, Glu 370, Gly 473 and Ile 371 (gp120) and between Arg59 (CD4) and Val430 (gp120)

Interfacial Cavities
1. Cavity 1 - largest (279A3) between concave middle of CD4 sheet and groove on gp120 2. Phe43 Cavity - smaller spherical (152A3) pocket in the gp120 that is plugged by Phe43 from CD4. It is at interface between inner and outer domains of gp120. even though there is little direct contact to CD4.
 * Lined by hydrophilic residues (1/2 from gp120, 1/2 from CD4)
 * Has water in cavity that serves as water buffer between gp120/cd4
 * gp120 residues that line cavity are sequence variable, surrounded by conserved residues.
 * Sub. the conserved residues==> affects CD4 binding
 * CD4 residues lining the cavity have only moderate effect on gp120 binding when mutated
 * Tolerance for variation in gp120 surface in this cavity creates an "anti-hotspot" that may help virus escape antibodies trying to block the CD4 binding site.
 * deeply buried in hydrophobic interior of gp120
 * Residues lining Phe43 cavity are hydrophobic, highly conserved, implying functional significance
 * Mutations at Thr 257 and Trp 427 (neither have contact/major contact) can reduce binding significantly

Changes in residues that line the cavity affect the binding of antibodies directed against the CD4-binding site

Antibody interface

 * 17b antibody - neutralizing, binds to CD-4 induced gp120 epitope, overlaps chemokine receptor-binding site
 * 17b epitope highly conserved
 * 17b contact surface acidic, hydrophobic contacts at the center
 * on gp120, it lies across base of bridging sheet
 * integrity of bridging sheet essential for 17b binding
 * gp120 basic, but only 1 salt bridge
 * no direct CD4-17b contacts
 * no gp120 residue contacts both 17b AND CD4
 * CD4 binds on opposite face of bridging sheet, providing contacts that stabilize conformation
 * Bridging sheet highly conserved

Figure 4

 * A. Fab 17b shown binding to gp120
 * B. Contact surface and V3 loop
 * C. Contact surface and V3 loop, turned 90 degrees to better show 17b epitope
 * D. Electrostatic potential at solvent-accessible surface 17b epitope surface more electropositive region. V3 loop is positively charged overall.
 * E. Cα-worm diagram of gp120

Chemokine-receptor site

 * 17b epitope overlaps with site where CCR5 interacts. Both highly conserved/induced by CD4 binding.
 * Hydrophobic and acidic surface of 17b heavy chain mimics Tyr-rich, acidic N-term of CCR5, important for gp120 binding/HIV-1 entry

Oligomer and gp41 interactions

 * gp120 most likely a trimeric complex with gp41 on the virion surface
 * N,C termini of full-length gp120 most important for interaction with gp41 gp
 * Expect gp41-interactive regions will extend away from core gp120 toward the viral membrane

Conformational change in core gp120

 * CD4 binding causes conformational gp120 change
 * Fab 17b binds to core gp120 only if CD4 is present
 * Phe43 cavity is between the inner/outer domain and the bridging sheet, essential to the structure

Figure 5

 * 1) Inner.outer domain shift occurs when CD4 binds with the formation of the Phe43 cavity
 * 2) Chemokine receptor binds to bridging sheet and the V3 loop, shifting the core gp120
 * 3) Further changes are triggered, leading to the fusion of viral and target cell membranes

Viral evasion of immune responses

 * 1) glycosylation and oligomeric occlusion hide the envelope protein surface from humoral immune response
 * 2) broadly neutralizing antibodies access 2 surfaces a surface that overlaps CD4-binding site that's shielded by the V1/V2 loop b surface that overlaps chemokine-receptor-binding site that's shielded by V2 and V3 loops
 * CD4-binding site recognition prevented by recession of binding pocket and topographical surface mismatch
 * Chemokine receptor region recognition prevented by:
 * 1) conformational change
 * 2) steric occlusion - blockage
 * 3) surface mismatch

Mechanistic implications for virus entry

 * Gp120 crucial for fusion of HIV to cell surface
 * simple process comprising viral oligomer and two host receptors.
 * Gp120 functions in positioning
 * 1) find viable host cell
 * 2) anchor virus to cell surface
 * 3) orient viral spike adjacent to target membrane
 * Gp120 function in timing
 * 1) hold gp41 in metastable comformation
 * 2) trigger/coordinate release of 3 N-term. fusion peptides of trimeric gp41


 * Entry initiated by binding of HIV-1 to cellular receptor CD4. Orients viral spike
 * CD4 binding induces conformational change in gp120, creating a metastable oligomer
 * results in movement of V2 loop,partially blocking V3 loop and CD4 interacting epitopes
 * creates/stabilizes bridging sheet
 * CD4 binding changes V3 region and alters exposure of V3 epitopes
 * Interaction of gp120-CD4 complex with chemokine receptor,Binding moves gp120 closer to target membrane

Crystallization

 * 1) Crystallize protein to cross-section of 30-40um
 * 2) Measure diffraction patterns with Bragg’s Law to determine space between electrons/atoms (2d model)
 * Originally crystals diffracted more than 2A, but were able to reduce the limit to 2.5A

Multiple Isomorphous Replacement

 * Electrons oscillate during diffraction, must phase them to find electron distribution (location) by inclusion of evenly spaced heavy metals/atom compounds


 * Tried over 20 different heavy-atom solutions


 * Isomorphism highest between K3IrCl6, and K2OsCl6 and Native form.

Molecular Replacement

 * Used Fourier Analysis (mathematical method) to determine 3d pattern of these heavy-atoms (Electron Density Model)
 * K3IrCl6 modelled as 9 partially occupied sites (2sites of occupancy)
 * Poor data quality, small isomorphous differences
 * K2OsCl6 (4 sites of occupancy), highest site at same as 2nd highest for K3IrCl6

Density Modification to Improve Electron Density Model

 * 1) Correlations in region internal to domain 1 of CD4 between experimental electron density and calculated model
 * 2) Linkage of unmodeled density (PRISM program)
 * 3) Recipricol-space averaging of the PRISM modeled density
 * 4) Real-space model subtraction (XPLOR)
 * 5) Solvent flattening - noise suppression to improve the quality of the phases and therefore the electron density
 * 6) Histogram matching- method in image processing of color adjustment of two images using the image histograms.It can be used to normalize two images.
 * 7) Negative-Density Truncation - refine the initial phases

Definitions

 * 1) epitope -That part of an antigenic molecule to which the T-cell receptor responds; a site on a large molecule against which an antibody will be produced and to which it will bind. biology-online.org dictionary
 * 2) chemokine - class of pro-inflammatory cytokines that have the ability to attract and activate leukocytes. They can be divided into at least three structural branches: c (chemokines, c), cc (chemokines, cc), and cxc (chemokines, cxc), according to variations in a shared cysteine motif. biology-online.org dictionary Small secreted proteins that stimulate chemotaxis of leucocytes. Chemokines can be subdivided into classes on the basis of conserved cysteine residues. The a -chemokines (IL-8, NAP-2, Gro- a, Gro- g , ENA-78 and GCP-2) have conserved C-X-C motif and are mainly chemotactic for neutrophils; the b -chemokines (MCP-1-5, MIP-1 a , MIP-1 b , eotaxin, RANTES) have adjacent cysteines (C-C) and attract monocytes, eosinophils or basophils; the g -chemokines have only one cysteine pair and are chemotactic for lymphocytes (lymphotactin), the d -chemokines are structurally rather different being membrane-anchored, have a C-X-X-X-C motif and are restricted (so far) to brain (neurotactin). Human genes for the a -chemokines are on Chr 4 and 10, for b -chemokines on Chr 17, for lymphotactin on Chr 1 and for neurotactin on Chr16. The receptors are G-protein coupled.Dictionary of Cell and Molecular Biology
 * 3) oligomeric spike/complex -
 * 4) trimeric spike/complex -
 * 5) fusogenic - Facilitating fusion of the viral envelope with the cellular plasma membrane. Nature Review Glossary
 * 6) anti-hotspot -
 * 7) cavity - a hollow place or space or a potential space, within the body or in one of its organs, it may be normal or pathological. biology-online.org dictionary
 * 8) linchpin - 1.A locking pin inserted in the end of a shaft, as in an axle, to prevent a wheel from slipping off. 2. A central cohesive element Farlex Dictionary
 * 9) humoral immune response - Those immune responses mediated by antibody. biology-online.org dictionary
 * 10) oligomeric occlusion - 1. The act of closure or state of being closed. 2. The relationship between all of the components of the masticatory system in normal function, dysfunction and parafunction.3. Momentary complete closure of some area in the vocal tract, causing stoppage of the breath and accumulation of pressure. biology-online.org dictionary
 * 11) glycocalyx - The region, seen by electron microscopy, external to the outer dense line of the plasma membranethat appears to be rich in glycosidic compounds such as proteoglycans and glycoproteins. Since these molecules are often integral membrane proteins and may be denatured by the processes of fixation for electron microscopy, it might be better to avoid the term or to refer to membrane glycoproteins or to proteoglycans associated with the cell surface. Dictionary of Cell and Molecular Biology
 * 12) ectodomain- is 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. In SARS-CoV the ectodomain of the spike protein is responsible for attachment to and entry into cells during infection. Wikipedia
 * 13) fab - fragment of immunoglobulin prepared by papain treatment. Fab fragments (45 kD) consist of one light chain linked through a disulphide bond to a portion of the heavy chain and contain one antigen binding Site. They can be considered as univalent antibodies. biology-online.org dictionary
 * 14) α-carbon trace -
 * 15) molecular replacement (technique) - is a method of solving the phase problem in X-ray crystallography. MR relies upon the existence of a previously solved protein structure which is homologous (similar) to our unknown structure from which the diffraction data is derived. Wikipedia
 * 16) isomorphous replacement (tech) -A method of determining diffraction phases from the differences in intensity between corresponding reflections from two or more isomorphous crystals. Most commonly used in the determination of protein structures, where it is possible to derive isomorphous crystals of native protein and of heavy-atom derivatives. Online Dictionary of Crystallography
 * 17) density modification (tech)-
 * 18) Accessible surface area -  is the surface area of a biomolecule (protein, DNA, etc.) that is accessible to a solvent.  It is used to "probe" the topography of a biomolecule, such as cavities, etc. Net-Sci

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