Biomedical subjects
Publications and source records attributed to I A Wilson.
Recent work has shown that large conformational changes in antibodies can accompany antigen binding. These structural changes can alter dramatically the size, shape and charge distribution in the antigen-binding pocket, and may present problems for accurate modeling of antibody structures.
During the past year, many new antibody structures have been determined, increasing our understanding of these immunologically important molecules. Of special interest are new catalytic antibodies, antibody-peptide and antibody-virus complexes, NMR structures, and structures illustrating conformational changes and antibody cross-reactivity.
Monoclonal anti-progesterone antibodies were raised by immunizing mice with progesterone coupled through either the C3, C6 or C11 positions to protein carrier (bovine serum albumin, BSA). The specificity of four antibodies for a range of steroids related to progesterone, some carrying substitutions at various ring positions, was studied by competitive inhibition in an ELISA system. The results demonstrated that the ring coupling position has a determining effect on the cross-reactivity of the antibodies obtained. The patterns of cross-reaction were interpreted in the light of the structure of the combining site of an anti-progesterone antibody (DB3) recently determined by X-ray crystallography, and inferences drawn about the orientation of steroid in the combining sites of the antibodies studied. Specifically, in two antibodies raised against progesterone-11-BSA, the orientation of steroid resembled that of the progesterone-DB3 complex, with positions C11 and C3 exposed and C6 and C20 buried; an antibody raised against progesterone-6-BSA bound steroid in an apparently similar disposition, except that C6 was exposed and C11 buried; finally, in an antibody raised against progesterone-3-BSA, all steroid positions other than C3 were apparently buried in the steroid-antibody complex.
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A new orthorhombic crystal form of Fab 17/9 has been determined in complex with a 7-mer peptide from influenza virus hemagglutinin (HA1 101-107, acetylated and amidated). The three-dimensional structure was resolved to 2.8 A with an improved refinement and better geometry than two previously determined Fab 17/9-peptide (HA1 100-108) complexes, facilitating a detailed description of the Fab-peptide interactions. The binding pockets and the peptide antigen are structurally similar in all three peptide complexes of Fab 17/9. The peptide adopts an extended conformation (residues 100 to 103) and a type I reverse turn (residues 104 to 107). Additionally, the antigenic determinant described here correlates well with previous epitope mapping studies. The structures of the free and antigen bound Fab illustrate the role of induced fit as a mechanism for antibody-antigen recognition. Fab 17/9 undergoes a large conformational change, mainly in the H3 loop, upon peptide binding. As a result, the shape of the binding pocket changes substantially in the liganded Fab. However, the backbone conformations of the other hypervariable loops (L2, L3, H1 and H2) show no significant difference between free and bound structures. The conformation of the L1 loop is also maintained in all structures, but its position relative to the framework varies in different crystal environments. The availability of three X-ray structures of an Fab-peptide complex in three different space groups makes it possible to clearly distinguish between crystal packing and antigen binding as the cause of structural differences. Two distinct H3-loop conformations, free and bound, are observed with no evidence otherwise for multiple conformations of the hypervariable loops (CDRs) or increased flexibility in either the free or bound forms.
Two major unanswered questions concerning the specificity of antibodies are: how do structurally different antigens bind with high affinity to the same antibody, and what are the limits of the antibody combining site complementarity and flexibility that contribute to such crossreactivity? We report here a comparative analysis of the X-ray structures of five conformationally different steroids in complex with the Fab' fragment of an anti-progesterone antibody DB3 at 2.7 A. This antibody is unable to complement completely the shape of the hydrophobic antigen so that crossreactivity occurs with other ligands without major structural rearrangements of the binding site. Antigen specificity can be explained through conserved interactions of DB3 with the steroid D-ring, whereas some of the crossreactivity is realized through different binding orientations of the steroid skeleton that place the A-ring into alternative pockets on the antibody surface. The restricted gene usage of the VGAM3.8 family in the generation of anti-progesterone monoclonal antibodies may be explained by the specific interaction of VH hallmark residues with the steroid D-ring. This first detailed structure of steroid interactions with a protein could be applied to the understanding of general mechanisms of steroid recognition as well as in the design of specific binding sites for small hydrophobic ligands.
BACKGROUND: Recent structural results have shown that antibodies use an induced fit mechanism to recognize and bind their antigens. Here we present the crystallographically determined structure of an Fab directed against an HIV-1 peptide (Fab 50.1) in the unliganded state and compare it with the peptide-bound structure. We perform a detailed analysis of the components that contribute to enhanced antigen binding and recognition. RESULTS: Induced fit of Fab 50.1 to its peptide antigen involves a substantial rearrangement of the third complementarity determining region loop of the heavy chain (H3), as well as a large rotation of the variable heavy (VH) chain relative to the variable light (VL) chain. Analysis of other Fab structures suggests that the extent of the surface area buried at the VL-VH interface correlates with the ability to alter antibody quaternary structure by reorientation of the VL-VH domains. CONCLUSION: Fab 50.1 exhibits the largest conformational changes yet observed in a single antibody. These can be attributed to the flexibility of the variable region. Comparisons of new data with previous examples lend to the general conclusion that a small VL-VH interface, due in part to a short H3 loop, permits substantial alterations to the antigen-binding pocket. This has major implications for the prediction, engineering and design of antibody-combining sites.
The crystal structure of the Fab fragment of a human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody Fab has been determined at 2.8 A resolution in complex with a linear 16-residue peptide from the third hypervariable region (V3) of gp120. The first 9 residues of the peptide are ordered in the electron density maps, and their conformation is in partial agreement with the beta-strand-type II beta-turn structure predicted for this portion of the V3 loop. Notably, several of the peptide residues that are well conserved among different HIV-1 isolates contact a nonpolar 25-A-long groove in the antibody-combining site. The largely extended structure of the peptide differs from the beta-turns seen as the primary determinants in other published anti-peptide Fab structures. Analysis of the specific Fab-peptide interactions only partially explains the MN isolate specificity shown by this antibody.
The monoclonal anti-progesterone antibody DB3 binds progesterone with nanomolar affinity (Ka approximately 10(9) M-1), suggesting high specificity. However, DB3 also cross-reacts with similar affinity with a subgroup of structurally distinct, progesterone-like steroids. Crystals of the unliganded Fab' and various steroid-Fab' complexes are isomorphous and belong to the hexagonal space group, P6(4)22, with unit cell dimensions of a = b = 135 A, c = 124 A. Structures of free and progesterone-bound Fab' have been determined by X-ray crystallography at 2.7 A resolution using molecular replacement techniques. Progesterone is bound in a hydrophobic pocket formed mainly by the interaction of three complementarity determining regions L1, H2 and H3. The orientation of the ligand in the binding site was aided by both crystallographic and biochemical analyses of substituted steroids. The indole side-chain of TrpH100 of the DB3 has two different conformations, inter-converting "open" and "closed" forms of the antibody combining site. The TrpH100 indole thus appears to be acting as an antibody-derived surrogate ligand for its own hydrophobic binding pocket. These structures provide the first atomic view of how a steroid interacts with a protein and offer a structural explanation for the restriction of the anti-progesterone response to the VGAM3.8 family of VH genes.
Phospholipase A2 (PLA2) from Indian cobra venom (Naja naja naja) was crystallized from ethanol in space group P4(3)2(1)2 in the presence of Ca2+. The x-ray crystal structure was determined to 2.3-A resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R value of 0.174 for 17,542 reflections between 6.0- and 2.3-A resolution (F > 2 sigma), including 148 water molecules. The 119-amino acid enzyme has an overall architecture strikingly similar to the other known PLA2 structures with regions implicated in catalysis showing the greatest structural conservation. Unexpectedly, three monomers were found to occupy the asymmetric unit and are oriented with their catalytic sites facing the pseudo-threefold axis with approximately 15% of the solvent accessible surface of each monomer buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms. The possible relevance of this unique trimeric structure is considered.
Three-dimensional structures of human and murine MHC class I molecules in complex with peptide antigens have begun to elucidate the mechanism of peptide binding. The MHC molecule has a highly conserved structure in which important variation occurs only in the residues lining the central polymorphic portion of the peptide binding cleft. Peptides bind in extended conformations deep within the cleft and have highly specific and conserved interactions at their amino and carboxyl termini and divergent interactions in the middle. Peptides longer than the canonical length for a given allele appear to bulge in the middle but maintain their interactions at their ends. One or more anchor residues are used by the peptide to establish tight and enduring presentation to T cells. Peptide-associated conformational differences in the MHC molecule itself may contribute to the T cell recognition process.
X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.
The atomic structure of glycinamide ribonucleotide transformylase, an essential enzyme in purine biosynthesis, has been determined at 3.0 A resolution. The last three C-terminal residues and a sequence stretch of 18 residues (residues 113 to 130) are not visible in the electron density map. The enzyme forms a dimer in the crystal structure. Each monomer is divided into two domains, which are connected by a central mainly parallel seven-stranded beta-sheet. The N-terminal domain contains a Rossmann type mononucleotide fold with a phosphate ion bound to the C-terminal end of the first beta-strand. A long narrow cleft stretches from the phosphate to a conserved aspartic acid, Asp144, which has been suggested as an active-site residue. The cleft is lined by a cluster of residues, which are conserved between bacterial, yeast, avian and human enzymes, and likely represents the binding pocket and active site of the enzyme. GAR Tfase binds a reduced folate cofactor and glycinamide ribonucleotide for the catalysis of one of the initial steps in purine biosynthesis. Folate analogs and multi-substrate inhibitors of the enzyme have antineoplastic effects and the structure determination of the unliganded enzyme and enzyme-inhibitor complexes will aid the development of anti-cancer drugs.
The x-ray structures of a murine MHC class I molecule (H-2Kb) were determined in complex with two different viral peptides, derived from the vesicular stomatitis virus nucleoprotein (52-59), VSV-8, and the Sendai virus nucleoprotein (324-332), SEV-9. The H-2Kb complexes were refined at 2.3 A for VSV-8 and 2.5 A for SEV-9. The structure of H-2Kb exhibits a high degree of similarity with human HLA class I, although the individual domains can have slightly altered dispositions. Both peptides bind in extended conformations with most of their surfaces buried in the H-2Kb binding groove. The nonamer peptide maintains the same amino- and carboxyl-terminal interactions as the octamer primarily by the insertion of a bulge in the center of an otherwise beta conformation. Most of the specific interactions are between side-chain atoms of H-2Kb and main-chain atoms of peptide. This binding scheme accounts in large part for the enormous diversity of peptide sequences that bind with high affinity to class I molecules. Small but significant conformational changes in H-2Kb are associated with peptide binding, and these synergistic movements may be an integral part of the T cell receptor recognition process.
Class I major histocompatibility complex (MHC) molecules interact with self and foreign peptides of diverse amino acid sequences yet exhibit distinct allele-specific selectivity for peptide binding. The structures of the peptide-binding specificity pockets (subsites) in the groove of murine H-2Kb as well as human histocompatibility antigen class I molecules have been analyzed. Deep but highly conserved pockets at each end of the groove bind the amino and carboxyl termini of peptide through extensive hydrogen bonding and, hence, dictate the orientation of peptide binding. A deep polymorphic pocket in the middle of the groove provides the chemical and structural complementarity for one of the peptide's anchor residues, thereby playing a major role in allele-specific peptide binding. Although one or two shallow pockets in the groove may also interact with specific peptide side chains, their role in the selection of peptide is minor. Thus, usage of a limited number of both deep and shallow pockets in multiple combinations appears to allow the binding of a broad range of peptides. This binding occurs with high affinity, primarily because of extensive interactions with the peptide backbone and the conserved hydrogen bonding network at both termini of the peptide. Interactions between the anchor residue (or residues) and the corresponding allele-specific pocket provide sufficient extra binding affinity not only to enhance specificity but also to endure the presentation of the peptide at the cell surface for recognition by T cells.
The three-dimensional structure of a specific antibody (Fab 17/9) to a peptide immunogen from influenza virus hemagglutinin [HA1(75-110)] and two independent crystal complexes of this antibody with bound peptide (TyrP100-LeuP108) have been determined by x-ray crystallographic techniques at 2.0 A, 2.9 A, and 3.1 A resolution, respectively. The nonapeptide antigen assumes a type I beta turn in the antibody combining site and interacts primarily with the Fab hypervariable loops L3, H2, and H3. Comparison of the bound and unbound Fab structures shows that a major rearrangement in the H3 loop accompanies antigen binding. This conformational change results in the creation of a binding pocket for the beta turn of the peptide, allowing TyrP105 to be accommodated. The conformation of the peptide bound to the antibody shows similarity to its cognate sequence in the HA1, suggesting a possible mechanism for the cross-reactivity of this Fab with monomeric hemagglutinin. The structures of the free and antigen bound antibodies demonstrate the flexibility of the antibody combining site and provide an example of induced fit as a mechanism for antibody-antigen recognition.
Glycosylated interleukin-2 (glyIL-2) has been crystallized in two crystal forms, and unglycosylated interleukin-2 (uIL-2) has been crystallized in three forms. The glycosylated form of the human recombinant IL-2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6(2)22 or its enantiomorph. The crystals diffract to 2.8 A and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0-5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL-2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5-5.6 in the monoclinic space group P2(1), and less frequently in the orthorhombic space group P2(1)2(1)2(1) from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross-seeding uIL-2 with seeds from hexagonal crystals of glyIL-2 promotes nucleation of trigonal crystals of unglycosylated IL-2. These trigonal crystals belong to the space group P3(1)21 or its enantiomorph, with similar cell dimensions to the glyIL-2 hexagonal crystals.
X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.