Abraham M. de Vos

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis.

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.

Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex

We have determined the 3.2 A X-ray crystal structure of the extracellular domain of the human epidermal growth factor receptor 2 (ErbB2 or HER2) in a complex with the antigen binding fragment of pertuzumab, an anti-ErbB2 monoclonal antibody also known as 2C4 or Omnitarg. Pertuzumab binds to ErbB2 near the center of domain II, sterically blocking a binding pocket necessary for receptor dimerization and signaling. The ErbB2-pertuzumab structure, combined with earlier mutagenesis data, defines the pertuzumab residues essential for ErbB2 interaction. To analyze the ErbB2 side of the interface, we have mutated a number of residues contacting pertuzumab and examined the effects of these mutations on pertuzumab binding and ErbB2-ErbB3 heterodimerization. We have also shown that conserved residues previously shown to be necessary for EGF receptor homodimerization may be dispensible for ErbB2-ErbB3 heterodimerization.

IL‐17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL‐17F, and implications for receptor binding

The proinflammatory cytokine interleukin 17 (IL‐17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL‐17 homolog, IL‐17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL‐6, IL‐8 and granulocyte colony‐stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL‐17F reveals that IL‐17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL‐17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL‐17s and neurotrophins with respect to receptor recognition.

Triggering Cell Death: The Crystal Structure of Apo2L/TRAIL in a Complex with Death Receptor 5

Formation of a complex between Apo2L (also called TRAIL) and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains. We report the crystal structure of the complex between Apo2L and the ectodomain of DR5. The structure shows three elongated receptors snuggled into long crevices between pairs of monomers of the homotrimeric ligand. The interface is divided into two distinct patches, one near the bottom of the complex close to the receptor cell surface and one near the top. Both patches contain residues that are critical for high-affinity binding. A comparison to the structure of the lymphotoxin-receptor complex suggests general principles of binding and specificity for ligand recognition in the TNF receptor superfamily.

VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 A resolution and mutational analysis of the interface.

BACKGROUND Vascular endothelial growth factor (VEGF) is a highly specific angiogenic growth factor; anti-angiogenic treatment through inhibition of receptor activation by VEGF might have important therapeutic applications in diseases such as diabetic retinopathy and cancer. A neutralizing anti-VEGF antibody shown to suppress tumor growth in an in vivo murine model has been used as the basis for production of a humanized version. RESULTS We present the crystal structure of the complex between VEGF and the Fab fragment of this humanized antibody, as well as a comprehensive alanine-scanning analysis of the contact residues on both sides of the interface. Although the VEGF residues critical for antibody binding are distinct from those important for high-affinity receptor binding, they occupy a common region on VEGF, demonstrating that the neutralizing effect of antibody binding results from steric blocking of VEGF-receptor interactions. Of the residues buried in the VEGF-Fab interface, only a small number are critical for high-affinity binding; the essential VEGF residues interact with those of the Fab fragment, generating a remarkable functional complementarity at the interface. CONCLUSIONS Our findings suggest that the character of antigen-antibody interfaces is similar to that of other protein-protein interfaces, such as ligand-receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface.

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.

BACKGROUND Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

Suppressor of Fused Regulates Gli Activity through a Dual Binding Mechanism

ABSTRACT The Hedgehog pathway drives proliferation and differentiation by activating the Gli/Ci family of zinc finger transcription factors. Gli/Ci proteins form Hedgehog signaling complexes with other signaling components, including the kinesin-like protein Costal-2, the serine-threonine kinase Fused, and Suppressor of Fused [Su(fu)]. In these complexes Gli/Ci proteins are regulated by cytoplasmic sequestration, phosphorylation, and proteolysis. Here we characterize structural and functional determinants of Su(fu) required for Gli regulation and show that Su(fu) contains at least two distinct domains: a highly conserved carboxy-terminal region required for binding to the amino-terminal ends of the Gli proteins and a unique amino-terminal domain that binds the carboxy-terminal tail of Gli1. While each domain is capable of binding to different Gli1 regions independently, interactions between Su(fu) and Gli1 at both sites are required for cytoplasmic tethering and repression of Gli1. Furthermore, we have solved the crystal structure of the amino-terminal domain of human Su(fu)27-268 at 2.65 Å resolution. This domain forms a concave pocket with a prominent acidic patch. Mutation at Asp159 in the acidic patch disrupts Gli1 tethering and repression while not strongly disrupting binding, indicating that the amino-terminal domain of Su(fu) likely impacts Gli binding through a mechanism distinct from that for tethering and repression. These studies provide a structural basis for understanding the function of Su(fu).

Crystal structure of a sweet tasting protein thaumatin I, at 1.65 Å resolution

The crystal structure of thaumatin I, a potently sweet protein isolated from the fruits of the West African shrub, Thaumatococcus danielli Benth, has been refined at a resolution better than 1.65 A using a combination of energy minimization and stereochemically restrained least-squares methods. The final model consists of all 207 amino acids, 28 alternate amino acid conformers and 236 waters, with a crystallographic R-factor of 0.145 for 19,877 reflections having F > 4 sigma F between 10.0 A and 1.65 A (R = 0.167 for all 24,022 reflections). The model has good stereochemistry, with root-mean-square deviations from ideal values for bond and angle distances of 0.014 A and 0.029 A, respectively. The estimated root-mean-square co-ordinate error is 0.15 A. The current model confirms the previously reported 3.1 A C alpha trace in both main chain connectivity and disulfide topology, including two disulfide bonds, that differed from the earlier reported biochemical determination. The structure contains three domains. The core of the molecule consists of an eleven-stranded, flattened beta-sandwich folded into two Greek key motifs. All beta-strands in this sandwich are antiparallel except the parallel N-terminal and the C-terminal strands. The average hydrogen bond length in this sandwich is 2.89 A, with an angle of 155.1 degrees. Two beta-bulges are found in one of the sheets. The second domain consists of two beta-strands forming a beta-ribbon and connected by an omega-loop, and contains a proline residue in cis conformation. This structural motif folds back against the main sandwich to form a smaller sandwich-like structure. The third domain is a disulfide-rich region stretching away from the sandwich portion of the molecule. It contains one alpha-helix and three short helical fragments. Two of the helical segments are connected by an unusually sharp turn, stabilized by a disulfide bridge. One of the three disulfide bonds in this domain takes on two conformations.

Receptor-selective Variants of Human Vascular Endothelial Growth Factor GENERATION AND CHARACTERIZATION

Vascular endothelial growth factor (VEGF) is a pleiotropic factor that exerts a multitude of biological effects through its interaction with two receptor tyrosine kinases,fms-like tyrosine kinase (Flt-1) or VEGF receptor 1 and kinase insert domain-containing receptor (KDR) or VEGF receptor 2. Whereas it is commonly accepted that KDR is responsible for the proliferative activities of VEGF, considerable controversy and uncertainty exist about the role of the individual receptors in eliciting many of the other effects. Based on a comprehensive mutational analysis of the receptor-binding site of VEGF, an Flt-1-selective variant was created containing four substitutions from the wild-type protein. This variant bound with wild-type affinity to Flt-1, was at least 470-fold reduced in binding to KDR, and had no activity in cell-based assays measuring autophosphorylation of KDR or proliferation of primary human vascular endothelial cells. Using a competitive phage display strategy, two KDR-selective variants were discovered with three and four changes from wild-type, respectively. Both variants had approximately wild-type affinity for KDR, were about 2000-fold reduced in binding to Flt-1, and showed activity comparable with the wild-type protein in KDR autophosphorylation and endothelial cell proliferation assays. These variants will serve as useful reagents in elucidating the roles of Flt-1 and KDR.

Vascular Endothelial Growth Factor–Induced Genes in Human Umbilical Vein Endothelial Cells: Relative Roles of KDR and Flt-1 Receptors

Objective—This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF. Methods and Results—We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A). We report that activation of KDR was sufficient to upregulate all the genes induced by VEGFwt. In contrast, there were no genes selectively upregulated by the Flt-selective mutant. However, high concentrations of the Flt-selective mutant could augment the expression of some genes induced by submaximal concentrations of VEGFwt but not the KDR-selective mutant. Conclusions—The binding of VEGF to its receptor, KDR, is necessary and sufficient to induce the gene expression profile induced by this growth factor. Furthermore, in human umbilical vein endothelial cells, the Flt-1 receptor appears to act as a decoy receptor, tempering the response to lower concentrations of VEGF.