J'aime C. Moehlman's Week 7

Terms and Definitions

 * 1) Syncytia:An epithelium or tissue characterized by cytoplasmic continuity, or a large mass of cytoplasm not separated into individual cells and containing many nuclei. Syncytia is the plural for syncytium.
 * 2) Fab fragments: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.
 * 3) X-ray Crystallography:The study of crystal structure using x-ray diffraction techniques.
 * 4) Tropism:An involuntary orienting response; positive or negative reaction to a stimulus source.
 * 5) Ternary (complex):term used to describe the tripartite combination of, for example, enzyme-cofactor-substrate or enzyme-substrate1-substrate2 for a multisubstrate enzyme, the active form involved in many enzyme-catalyzed reactions.
 * 6) Ramachandran:A graphical representation in which the dihedral angle of rotation about the alpha-carbon to carbonyl-carbon bond in polypeptides is plotted against the dihedral angle of rotation about the alpha-carbon to nitrogen bond.
 * 7) Isomorphously: The similarities between two forms of a structure in an organism.
 * 8) Hydrazone:a class of organic compounds with the structure R2C=NNR2. They are related to ketones and aldehydes by the replacement of the oxygen with the NNR2 functional group. They are formed usually by the action of hydrazine on ketones or aldehydes.
 * 9) Annealing:1. Toughening upon slow cooling. 2. Used in the context of dna renaturation after temperature dissociation of the two strands. Rate of annealing is a function of complementarity. 3. Fusion of microtubules or microfilaments end to end.
 * 10) Canonical:Classical, archetypal or prototypic.
 * 11) Torsion angles:The twisting/ changing of something about its axis; the angle is how far it rotated.

Definitions from:
 * biology-online.org dictionary
 * Dictionary of Cell and Molecular Biology
 * The Free Dictionary

Dual conformations for the HIV-1 gp120 V3 loop in complexes with different neutralizing Fabs

 * Stanfield et al. (1999)

Introduction

 * HIV-1 is a member of the lentivirus subfamily of retroviruses.
 * Beta- chemokine receptors have been implicated as the viral secondary receptors.
 * CXCR4 acrs as the primary receptor for T cell tropic SI isolates.
 * The V3 region is a disulfide look of about 40 amino acids.
 * The V3 region is one of the major immunogenic sites.
 * Exposure of the V3 loop to the gp120 protein varies depending on the viral isolate type and increases during interactions with CD4.
 * Sequence changes in V3 can alter ciral cell tropism, antibody neutralization, neutralization of soluble CD4, syncytium formation, and chemokine receptor usage.
 * T- tropic V3 sequences are usually more basic in charge than M-tropic sequences through accumulation of positively charged residues.
 * Some amino acid positions in the loop are highly variable in amino acid composition.
 * Their investigation was conducted by studying neutralizing antibodies and their complexes with V3 peptides to determine the tertiary conformation of the V3 loop.
 * The researchers believe that they can explain coreceptor usage and the changes that take place in the virus upon conversion from a primary M-tropic isolate to the T-tropic strains associated with disease progression.
 * Fab 50.1 and 59.1 are the two key structures of antibody fab fragments:
 * Fab 50.1 is thought to be highly specific for the MN viral strain
 * Fab 59.1 is thought to strongly neutralize IIIB and weakly neutralizes MN.
 * In the 59.1 peptide complex 10 amino acid residues were visible and five of these residues had structural counterparts in the 50.1 complex.
 * The sidechain of the Arg residue is bound in a deep, negatively charged pocket on the antibody surface.
 * Torsion angles are discussed throughout their research.
 * They used a synthetic hydrogen bond to mimic incorporating a hydrazone bond-- designed to replace CO-HN hydrogen bond.
 * The Fab 58.2 crystal structure shows that it is a highly neutralizing antibody that neutralizes both T-tropic and M-tropic viral strains.

Results and Discussion
Structure determination and refinement
 * The crystal structures for Fab 58.2 in complex with one linear and two hydrazone- linked cyclic peptide have been determined to 2.0 A and 2.8 A.
 * Structures were determined by the molecular replacement method using previously determined Fab structures.
 * All structures were refined with the X-PLOR simulated annealing refinement protocol.
 * Analysis of the Ramachandran plot by PROCHECK shows that 89% of the residues are in most favored positions with three residues in disallowed regions.
 * Disallowed residues include two residues in a disordered loop in the constant heavy chain which almost always displays high displacement parameters in other antibody structure determinations.
 * The Ser loop complex has 87% of the residues in most favored regions with five residues in disallowed regions.

Structure description and comparisons
 * Fab 58.2 is a mouse antibody, the CDR loops L1, L2, L3, and H2 belong to canonical classes 5,1,1, and 1.
 * The linear Aib142 peptide has an extended conformation for residues RIHI, which connect to a type I turn around residues GPGR.
 * The Fab uses five of its six CDR loops to bind peptide; CDR L2 is not used and CDR H1 makes only minor contacts.
 * A total of 124, 103, and 77 van der Waals contacts are made between the Fab and peptide fir the Aib142.
 * The resolution of the His loop and Ser loop structures is insufficient to allow accurate placement of ordered water molecules, one strong peak of density in the Fab binding site of all three structures was assigned as a water position.
 * The epitope specificity for 58.2 was determined previously by screening a peptide display libray expressing 1.5*10^8 unique 20 amino acid peptide sequences.
 * The epitope mapping results showed what epitopes could be replaced with amino acids, and which they could not be.
 * These epitopes also helped with specific things such as; antibody binding and determining the conformation of the tip of the V3 loop.

Two different V3 conformations
 * The most notable feature of the V3 peptide conformation while bound to Fab 58.2 is that it differs from the two previously determined structures for the same or similar peptide in complex with two other Fabs.
 * The three antibodies were all generated against the same peptide immunogen RP70.
 * The two peptides bount to Fabs 50.1 and 59.1 have torsion angles for a type II- Beta turn around residues GPGR.
 * It was not expected that the peptide bound to Fab 58.2 had torsion angles for a type I-beta turn around for the same residues.
 * The mainchain torsinon angles for these four residues differ significantly between the two peptide-conformational types.
 * The peptides bound to Fabs 59.1 and 58.2 both contain a Beta turn followed by a double bend, but the different torsion angles for the central GPGR residues change the precise character of these turns and bends.
 * NMR studies have supported the proposal that glycosylation of the V3 peptide can affect its conformation and that sequence changes affect the conformation.

Correlation of structure with function
 * We clearly observe two different conformations for the V3 loop even when bound to neutralizing antibodies.
 * The V3 loop conformations are biologically relevant because as the antibodies bind to viral gp120 to effect the neutralization activity.
 * These conformations may just show that the V3 loop is flexible and able to adopt different conformations.
 * The only reported crystals of gp120 to date have been obtained for protein where this loop was truncated at its base-- the crystals of gp120 with the loop intact loop haven't yet been reported.

Biological Implications

 * The V3 region of gp120 is involved in many aspects of virus infectivity.
 * The high consevation of Gly-Pro-Gly-Arg/Gln motif at the tip of the gp120 V3 loop suggests that structural conservation is related to biological function.
 * Studies have shown that altering one or more amino acid residues within the stem of the V3 loop leads to changes in viral tropism.
 * Further study could lead to small molecule drug design based on its 3-D structure, or vaccine development through the stabilization of biologically relevant conformations.

Materials and Methods

 * Aib142 was prepared by chemical synthesis using N(alpha)-9-fluorenylmethoxyl-carbonyl protected amino acids and Fmoc-Rink's amide resin on an Advance Chemtech ACT350 peptide synthesizer.
 * The N terminus was acetylated and the sidechain of Cys was protected with an acetamidomethyl group.
 * Trifluoroacetic acid was used to cleave the peptide from the resin.
 * Cyclic peptides were stnthesized by sold phase stnthesis using modified amino acids to form the hydrazone link; then purified and confirmed by mass spectrometry.
 * Space groups and unit cell dimensions for all crystal forms were determined by precession photography.
 * The Fab 58.2/Ser loop complex was determined by using the X-PLOR rotation function and PC refinement.
 * To estimate the elbow angle for the Fab 58.2- Aib142 structure, the Crowther Fast Rotation Function, was used to carry out cross-rotation functions with over 100 available Fab coordinate sets
 * Structure determination was done by PC refinement and translation function from X-PLOR

Figures and Tables

 * Figure 1:Shows the sequences of the V3 loop peptides used in crystallization of the peptide-Fab complexes. Fabs 58.2, 50.1, and 59.1 were raised against the 40 amino acid, disulfide linked peptide RP70, which has the MN viral sequence. The disulfide bonds linked the two cysteine residues. The Aib residue in peptide Aib142 replaces an Ala residue in the original sequence.
 * Figure 2:(a)shows the Fab 58.2-Aib142 complex; there are similarities among each pairing. (b)The Fab 58.2-His loop complex.Some of the atoms are labeled on the chains. (c)The Fab 58.2-Ser loop complex shows two different views of the structures.
 * Figure 3:This figure shows the comparison of the conformation of the H1 loops of Fab 58.2, N10, and AN02.
 * Figure 4:This figure shows the electron density for the V3 peptides bound to Fab 58.2.(a)shows the electron density for Fab 58.2-Aib142 complex- 11 of 24 residues are visible. (b) the electron density for the stimulating annealing shows the region around residues for RAibFY. (c) SA density for the His loop peptide. (d)SA density for the Ser loop peptide; in the figure the hydrazone linker was not included in this model due to weak electron density.
 * Figure 5:Shows Fab 58.2 binding pocket bound to the Aib142 peptide. (a)This is a representation of the antibody combining site with Aib142 peptide. (b) This is the stereoview of the combining site; shows the peptide, contacting residues, and both the light and heavy chain.
 * Figure 6:This figure shows the dual conformations of the V3 loop. It shows superimposed peptides bound to Fab 50.1, 59.1, and 58.2; they are superimposed based off of their torsion angles using the C(alpha) atoms.
 * Table 1:The table showed the x-ray diffraction data and refinement statistics for Fab 58.2 complexes. This table essentially hows all the data discovered based on everything for Fab 58.2.
 * Table 2:This table shows the data discovered from the van der Waals contacts between the Fab 58.2 and bound peptides; it shows the radii standard. The Fab residues contacted in all complexes are shown, and then other specific complexes.
 * Table 3:This table shows the hydrogen bonds and sal bridge interactions; here we can see how each peptide atom differs from one another.
 * Table 4:This table shows the residues with similar torsion angles bound to the 3 different Fabs.

Group Journal Club
[[Media:Protein Structure Journal Club Presentation.ppt| Protein Structure Journal Club]]