Adolfo H. Moraes

Solution and high-pressure NMR studies of the structure, dynamics, and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

Beta‐parvalbumins from different fish species have been identified as the main elicitors of IgE‐mediated reactions in fish‐allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross‐reactivity among fish parvalbumins. In addition, the high‐pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca2+‐free forms for potential use in immunotherapy. Proteins 2014; 82:3032–3042.

Epitope mapping by solution NMR spectroscopy

Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X‐ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations. Copyright

Structural basis for the dissociation of α-synuclein fibrils triggered by pressure perturbation of the hydrophobic core.

Parkinson’s disease is a neurological disease in which aggregated forms of the α-synuclein (α-syn) protein are found. We used high hydrostatic pressure (HHP) coupled with NMR spectroscopy to study the dissociation of α-syn fibril into monomers and evaluate their structural and dynamic properties. Different dynamic properties in the non-amyloid-β component (NAC), which constitutes the Greek-key hydrophobic core, and in the acidic C-terminal region of the protein were identified by HHP NMR spectroscopy. In addition, solid-state NMR revealed subtle differences in the HHP-disturbed fibril core, providing clues to how these species contribute to seeding α-syn aggregation. These findings show how pressure can populate so far undetected α-syn species, and they lay out a roadmap for fibril dissociation via pathways not previously observed using other approaches. Pressure perturbs the cavity-prone hydrophobic core of the fibrils by pushing water inward, thereby inducing the dissociation into monomers. Our study offers the molecular details of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. Understanding the molecular forces behind the formation of pathogenic fibrils uncovered by pressure perturbation will aid in the development of new therapeutics against Parkinson’s disease.

A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope

Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

An Overview on Protein Structure Determination by NMR: Historical and Future Perspectives of the use of Distance Geometry Methods

Determination of the protein high-resolution structures is essential for the understanding of complex biological mechanisms, for the development of biotechnological methods, and for other applications such as drug discovery. Protein structures solved by nuclear magnetic resonance (NMR) rely on a set of semiquantitative short-range distances and angles information. The exploration of the whole conformational space imposed by the experimental restraints is not a computationally simple problem. The lack of precise distances and angles does not allow to find solutions to this problem by fast geometric algorithms. The main idea is to define an atomic model for the protein structure and to exploit all known geometric angle and distance information along with the semi-quantitative short-range experimental information from NMR. We give an overview of the development of computational methods aimed at solving the problem either by metric matrix distance geometry or using other methods such as simulated annealing. We also discuss future demands and perspectives for structural calculations using NMR data. The need of determining larger and more complex protein structures implies the strong necessity of developing new methods for structural calculation with sparse data.

1H, 13C and 15N resonance assignments and second structure information of Gad m 1: a β-parvalbumin allergen from Atlantic cod (Gadus morhua)

Gad m 1 is the major allergen from Atlantic cod. It belongs to β-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. β-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of β-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a β-parvalbumin: Gad m 1. This report includes: 1H, 13C and 15N resonance assignments of Gad m 1 as well as the second structure information based on the 13C chemical shifts.

Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca 2+ -binding affinity in a C-domain mutant of cardiac troponin C

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

Antibody Binding Modulates Conformational Exchange in Domain III of Dengue Virus E Protein

ABSTRACT Domain III of dengue virus E protein (DIII) participates in the recognition of cell receptors and in structural rearrangements required for membrane fusion and ultimately viral infection; furthermore, it contains epitopes for neutralizing antibodies and has been considered a potential vaccination agent. In this work, we addressed various structural aspects of DIII and their relevance for both the dengue virus infection mechanism and antibody recognition. We provided a dynamic description of DIII at physiological and endosomal pHs and in complex with the neutralizing human antibody DV32.6. We observed conformational exchange in the isolated DIII, in regions important for the packing of E protein dimers on the virus surface. This conformational diversity is likely to facilitate the partial detachment of DIII from the other E protein domains, which is required to achieve fusion to the host cellular membranes and to expose the epitopes of many anti-DIII antibodies. A comparison of DIII of two dengue virus serotypes revealed many common features but also some possibly unexpected differences. Antibody binding to DIII of dengue virus serotype 4 attenuated the conformational exchange in the epitope region but, surprisingly, generated exchange in other parts of DIII through allosteric effects. IMPORTANCE Many studies have provided extensive structural information on the E protein and particularly on DIII, also in complex with antibodies. However, there is very scarce information regarding the molecular dynamics of DIII, and almost nothing is available on the dynamic effect of antibody binding, especially at the quantitative level. This work provides one of the very rare descriptions of the effect of antibody binding on antigen dynamics.

1H, 13C and 15N resonance assignments and second structure information of Fag s 1: Fagales allergen from Fagus sylvatica.

AbstractFagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the 1H, 15N and 13C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.

Solution and high-pressure NMR studies of the structure, dynamics and stability of the cross-reactive allergenic cod parvalbumin Gad m 1

Background IgE-mediated allergy to fish is a frequent cause of lifethreatening allergic reactions. Allergen-specific immunotherapeutic approaches often make use of modified proteins that were designed to be hypoallergenic to reduce the risk of reactions. Since allergen-specific immunotherapy is the only treatment that provides long-term clinical benefits, determining the structure and molecular dynamics characteristics of allergens is necessary to understand how they interact with IgE and how to interfere with it by modifying the IgE-binding sites. b-parvalbumins represent one of the largest animal food-allergen families and are considered cross-reactive pan-allergens in fish. The b-parvalbumin structure is characterized by the presence of three EF-hand motifs (helix-loop-helix) called AB, CD and EF, but only CD and EF can chelate calcium ions. In order to contribute to the understanding of the allergenicity and the importance of the Ca2+-binding motifs for the stability of b-parvalbumins, we studied the major allergen of cod (Gadus morhua), Gad m 1, a member of the parvalbumin protein family