TRPA1 gene variants hurting our feelings.

TRPA1 is a Ca2+-permeable, non-selective cation channel that is activated by thermal and mechanical stimuli, an amazing variety of potentially noxious chemicals, and by endogenous molecules that signal tissue injury. The expression of this channel in nociceptive neurons and epithelial cells puts it at the first line of defense and makes it a key determinant of adaptive protective behaviors. For the same reasons, TRPA1 is implicated in a wide variety of disease conditions, such as acute, neuropathic, and inflammatory pains, and is postulated to be a target for therapeutic interventions against acquired diseases featuring aberrant sensory functions. The human TRPA1 gene can bare mutations that have been associated with painful conditions, such as the N855S that relates to the rare familial episodic pain syndrome, or others that have been linked to altered chemosensation in humans. Here, we review the current knowledge on this field, re-evaluating some available functional data, and pointing out the aspects that in our opinion require attention in future research. We make emphasis in that, although the availability of the human TRPA1 structure provides a unique opportunity for further developments, far more classical functional studies using electrophysiology and analysis of channel gating are also required to understand the structure-function relationship of this intriguing channel.

A nucleotide-switch mechanism mediates opposing catalytic activities of Rel enzymes.

Bifunctional Rel stringent factors, the most abundant class of RelA/SpoT homologs, are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3' of guanosine tri-/diphosphate (GTP/GDP) to synthesize the bacterial alarmone (p)ppGpp, and also catalyze the 3' pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide-switch mechanism controls catalysis by Thermus thermophilus Rel (RelTt). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of RelTt (RelTtNTD) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation.

Structural insights into serine protease inhibition by a marine invertebrate BPTI Kunitz-type inhibitor.

Proteins isolated from marine invertebrates are frequently characterized by exceptional structural and functional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactyla helianthus, displays activity not only against serine-, but also against cysteine-, and aspartate proteases. As an initial step to evaluate the molecular basis of its activities, we describe the crystallographic structure of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7Å resolution. The overall structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-like Kunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitz inhibitor complexes. However, a prominent stabilizing role within the interface was attributed to arginine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinity against trypsin, if the P3 residue of ShPI-1 is mutated to alanine. Together with the increased role of Arg(11) at P3 position, slightly reduced interactions at the prime side (Pn') of the primary binding loop and at the secondary binding loop of ShPI-1 were detected. In addition, the structure provides important information for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnological applications.

Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site.

The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.

Switching on cytotoxicity by a single mutation at the heavy chain variable region of an anti-ganglioside antibody.

Gangliosides are sialic acid-containing glycosphingolipids present in the plasma membrane of most mammalian cells. In humans, the expression of the N-glycolylated (Neu5Gc) variant of the sialic acid has been associated with malignant transformation, constituting therefore an attractive target for cancer immunotherapy. P3 monoclonal antibody (mAb) recognizes Neu5Gc-containing gangliosides, as well as sulfatides. Heavy chain CDR3 (H-CDR3) arginine residues have been shown to be crucial for ganglioside recognition, but less important for anti-idiotypic antibody binding. Here, we describe the effect on antibody reactivity of different mutations involving a single H-CDR3 acid residue. Substitution of glutamate 99 (Kabat numbering) by arginine, aspartate or serine residues resulted in no differences in anti-idiotype binding. However, the first mutation caused increased reactivity with the antigen, including a cytotoxic effect of the antibody on ganglioside-expressing cells previously unseen for the wild type antibody. Another antibody that recognizes N-glycolyl-GM3 ganglioside (GM3(Neu5Gc)), but not other glycolipids, named 14F7, exhibits also an arginine-enriched H-CDR3 and a complement-independent cell death activity. Unlike 14F7 mAb, the cytotoxicity of the P3 E(99)→R mutant antibody did not exclusively depend on ganglioside expression on tumor cells.

Crystal structure of an anti-ganglioside antibody, and modelling of the functional mimicry of its NeuGc-GM3 antigen by an anti-idiotypic antibody.

N-Glycolylated (NeuGc) gangliosides are tumor-specific antigens and as such represent attractive targets for cancer immunotherapy. The chimeric antibody chP3 selectively recognizes a broad variety of NeuGc gangliosides, showing no cross-reactivity to the highly similar N-acetylated (NeuAc) gangliosides that are common cellular antigens in humans. Here, we report the crystal structure of the chP3 Fab and its computer-docking model with the trisaccharide NeuGcalpha3Galbeta4Glcbeta, which represents the carbohydrate moiety of the tumor-antigen NeuGc-GM3. The interaction involves only the heavy chain of the chP3 antibody. The modelled complex is consistent with all available experimental data and shows good surface complementarity. The negatively charged sialic acid residue NeuGc is buried in a pocket flanked by two arginine residues, VH Arg31 and VH Arg100A. We have further investigated the interaction of chP3 with its anti-idiotypic antibody, 1E10 (also known as Racotumomab), currently in clinical trials as a cancer vaccine. While many of the chP3 residues predicted to interact with the NeuGc ganglioside also feature prominently in the modelled complex of chP3 and 1E10, we do not observe structural mimicry. Rather, we suspect that the anti-idiotype 1E10 may serve as an imprint of the structural characteristics of the chP3 idiotype and, consequently, give rise to antibodies with P3-like properties upon immunization.

Nimotuzumab, an antitumor antibody that targets the epidermal growth factor receptor, blocks ligand binding while permitting the active receptor conformation.

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) in cancer cells correlates with tumor malignancy and poor prognosis for cancer patients. For this reason, the EGFR has become one of the main targets of anticancer therapies. Structural data obtained in the last few years have revealed the molecular mechanism for ligand-induced EGFR dimerization and subsequent signal transduction, and also how this signal is blocked by either monoclonal antibodies or small molecules. Nimotuzumab (also known as h-R3) is a humanized antibody that targets the EGFR and has been successful in the clinics. In this work, we report the crystal structure of the Fab fragment of Nimotuzumab, revealing some unique structural features in the heavy variable domain. Furthermore, competition assays show that Nimotuzumab binds to domain III of the extracellular region of the EGFR, within an area that overlaps with both the surface patch recognized by Cetuximab (another anti-EGFR antibody) and the binding site for EGF. A computer model of the Nimotuzumab-EGFR complex, constructed by docking and molecular dynamics simulations and supported by mutagenesis studies, unveils a novel mechanism of action, with Nimotuzumab blocking EGF binding while still allowing the receptor to adopt its active conformation, hence warranting a basal level of signaling.

Gangliosides, Ab1 and Ab2 antibodies II. Light versus heavy chain: An idiotype-anti-idiotype case study.

The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.

Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside.

Phage display technology makes it possible to introduce and rapidly screen diversity in antibody binding sites. Chain shuffling has been successfully used to humanize murine antibody fragments and also to obtain affinity matured variants. Here we report a different application of this method: the use of chain shuffling to overcome improper prokaryotic expression behavior of a hybridoma-derived single-chain antibody fragment. Construction and expression of such recombinant antibody fragments remain as empirical entities, hampered by the inability to express some antibody genes coming from eukaryotic cells in bacterial expression systems. Such problems are different for each combination of variable regions and can be serious enough to preclude the use of some hybridomas as sources of V regions to obtain recombinant antibody fragments. The particular binding properties and potential usefulness of some monoclonal antibodies make it highly desirable to bypass these technical limitations in order to develop smaller size therapeutic agents in the form of antibody fragments. The 14F7 mouse monoclonal antibody is one such attractive candidate due to its high specificity for the N-glycolyl GM3 ganglioside overexpressed in tumor cells and its ability to distinguish this antigen from closely related gangliosides like N-acetyl GM3. Our goal was to construct a phage-displayed single-chain Fv antibody fragment derived from 14F7. After cloning the original variable regions from the 14F7 hybridoma in a phagemid vector, we were unable to detect either binding activity or even expression of antibody fragments in bacteria, despite repetitive efforts. We constructed light-chain shuffling libraries, from which functional antibody fragments were readily selected. These combined the original 14F7 heavy chain variable region with a wide variety of unrelated murine and human light-chain variable regions. New antibody fragments retained the valuable properties of the monoclonal antibody in terms of fine specificity, affinity and tumor recognition. They were readily produced by bacteria, either in phage-displayed form or as soluble molecules, and provided a panel of potentially useful variants for cancer diagnosis and immunotherapy. Chain shuffling and phage display were found to be useful strategies for selecting antibody fragments on the basis of both prokaryotic expression and antigen binding criteria.

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3.

N-glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 A resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcalpha3Galbeta4Glcbeta. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.