Yibin Xu

Antibodies specifically targeting a locally misfolded region of tumor associated EGFR

Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR287–302 complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFRC271A/C283A mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFRC271A/C283A. Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.

Crystal structure of the entire ectodomain of gp130: insights into the molecular assembly of the tall cytokine receptor complexes.

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1–6, D1–D6) at 3.6 Å resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4–D6) at 1.9 Å. This represents the first atomic resolution structure of the complete ectodomain of any “tall” cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.

SPRY domain-containing SOCS box protein 2: crystal structure and residues critical for protein binding.

The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent beta-sandwich, consisting of two seven-stranded beta-sheets wrapped around a long loop that extends from the centre strands of the inner or concave beta-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.

Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.

Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.

Crystal structure of PfRh5, an essential P. falciparum ligand for invasion of human erythrocytes

Plasmodium falciparum causes the most severe form of malaria in humans and is responsible for over 700,000 deaths annually. It is an obligate intracellular parasite and invades erythrocytes where it grows in a relatively protected niche. Invasion of erythrocytes is essential for parasite survival and this involves interplay of multiple protein–protein interactions. One of the most important interactions is binding of parasite invasion ligand families EBLs and PfRhs to host receptors on the surface of erythrocytes. PfRh5 is the only essential invasion ligand within the PfRh family and is an important vaccine candidate. PfRh5 binds the host receptor basigin. In this study, we have determined the crystal structure of PfRh5 using diffraction data to 2.18 Å resolution. PfRh5 exhibits a novel fold, comprising nine mostly anti-parallel α-helices encasing an N-terminal β-hairpin, with the overall shape being an elliptical disk. This is the first three-dimensional structure determined for the PfRh family of proteins. DOI: http://dx.doi.org/10.7554/eLife.04187.001

Production of a human neutralizing monoclonal antibody and its crystal structure in complex with ectodomain 3 of the interleukin-13 receptor α1.

Gene deletion studies in mice have revealed critical roles for IL (interleukin)-4 and -13 in asthma development, with the latter controlling lung airways resistance and mucus secretion. We have now developed human neutralizing monoclonal antibodies against human IL-13Rα1 (IL-13 receptor α1) subunit that prevent activation of the receptor complex by both IL-4 and IL-13. We describe the crystal structures of the Fab fragment of antibody 10G5H6 alone and in complex with D3 (ectodomain 3) of IL-13Rα1. Although the structure showed significant domain swapping within a D3 dimer, we showed that Arg(230), Phe(233), Tyr(250), Gln(252) and Leu(293) in each D3 monomer and Ser(32), Asn(102) and Trp(103) in 10G5H6 Fab are the key interacting residues at the interface of the 10G5H6 Fab-D3 complex. One of the most striking contacts is the insertion of the ligand-contacting residue Leu(293) of D3 into a deep pocket on the surface of 10G5H6 Fab, and this appears to be a central determinant of the high binding affinity and neutralizing activity of the antibody.

LRIG1 Extracellular Domain: Structure and Function Analysis.

We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR.

The signalling conformation of the insulin receptor ectodomain

Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin’s negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.The insulin receptor plays a key role in many physiological processes, yet how insulin effects receptor signaling at the structural level remains incomplete. Here the authors present a high-resolution cryo-EM structure of a high-affinity form of the insulin-bound insulin receptor ectodomain that sheds light on the mechanism of signal transduction.

How ligand binds to the insulin-like growth factor receptor

The human insulin and type 1 insulin-like growth factor receptor are homologous receptor tyrosine kinases. They are formed as disulphide-linked homodimers and share 58% sequence identity. The type 1 insulin-like growth factor receptor (IGF-1R) is involved in normal human growth and development. Aberrant IGF-1R signalling is implicated in cancer proliferation and metastasis and the receptor hence has undergone extensive investigation as a potential anti-cancer target. Insulin-like growth factor binding is understood to relax conformational restraints within the homodimer, initiating trans-phosphorylation of the receptor tyrosine kinase domains. Our earlier crystallographic studies have focused on the insulin receptor [1]. However, there are no three-dimensional structural data for the intact IGF-1R ectodomain that might inform atomic-level understanding of how insulin-like growth factors (i.e., IGF-1 and IGF-2) bind to this recptor. To resolve these issues, we present the first and landmark crystal structures of the intact IGF-1R ectodomain —in both apoand IGF-1 bound form, refined using data to 3.2 and 3.4 Å resolution, respectively (see images below). In addition to providing a wealth of atomic detail, these structures lead us to suggest that the way in which ligand binds is fundamentally different to the paradigm that has been in place for a number of decades.