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Abstract: Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc∊RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc∊RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc∊RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

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Abstract: The transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.

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Abstract: X‐ray structures of homopolymeric human L‐ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa‐nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ 3 ‐oxo)tris[(μ 2 ‐glutamato‐κO:κO′)](glutamato‐κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the level of the protein surface, capable of contributing coordination bonds and electrostatic guidance.

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Abstract: The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells1,2. Upon binding to genomic lesions, these enzymes utilise NAD+ to modify a plethora of proteins with mono- and poly(ADP-ribose) signals that are important for subsequent chromatin decompaction and repair factor recruitment3,4. These post-translational modification events are predominantly serine-linked and require HPF1, an accessory factor that is specific for the DNA damage response and switches the amino-acid specificity of PARP1/2 from aspartate/glutamate to serine residues5–10. Here, we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from both PARP1/2 and HPF1. We further show that the assembly of this new catalytic centre is essential for DNA damage-induced protein ADP-ribosylation in human cells. In response to DNA damage and NAD+ binding site occupancy, the HPF1–PARP1/2 interaction is enhanced via allosteric networks operating within PARP1/2, providing an additional level of regulation in DNA repair induction. As HPF1 forms a joint active site with PARP1/2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors.

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Abstract: Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-β-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the only enzyme responsible for this modification via the addition of an α-1,6-linked fucose to N-glycans. To date, the details of substrate recognition and catalysis by FUT8 remain unknown. Here, we report the crystal structure of FUT8 complexed with GDP and a biantennary complex N-glycan (G0), which provides insight into both substrate recognition and catalysis. FUT8 follows an SN2 mechanism and deploys a series of loops and an α-helix which all contribute in forming the binding site. An exosite, formed by one of these loops and an SH3 domain, is responsible for the recognition of branched sugars, making contacts specifically to the α1,3 arm GlcNAc, a feature required for catalysis. This information serves as a framework for inhibitor design, and helps to assess its potential as a therapeutic target.