Philipp Neudecker

Structure of an Intermediate State in Protein Folding and Aggregation

Protein Tipping Point Amyloid fibrils are insoluble protein aggregates that play a role in various degenerative diseases. Recent experiments have provided insight into fibrillar structures; however, the mechanisms of aggregation remain unclear. Neudecker et al. (p. 362; see the Perspective by Eliezer) report the structure of a transient folding intermediate in a protein SH3 domain known to undergo aggregation. The intermediate is stabilized by non-native interactions and exposes an aggregation-prone β strand. Thus, for this protein, folding from the intermediate state will compete with aggregation. A folding intermediate of a protein SH3 domain is prone to aggregation, which competes with native folding. Protein-folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used relaxation dispersion nuclear magnetic resonance spectroscopy to determine the structure of a low-populated, on-pathway folding intermediate of the A39V/N53P/V55L (A, Ala; V, Val; N, Asn; P, Pro; L, Leu) Fyn SH3 domain. The carboxyl terminus remains disordered in this intermediate, thereby exposing the aggregation-prone amino-terminal β strand. Accordingly, mutants lacking the carboxyl terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form fibrillar aggregates. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.

Structural Coupling of SH2-Kinase Domains Links Fes and Abl Substrate Recognition and Kinase Activation

Summary The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase αC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Probing chemical shifts of invisible states of proteins with relaxation dispersion NMR spectroscopy: how well can we do?

Carr-Purcell-Meiboom-Gill relaxation dispersion NMR spectroscopy has evolved into a powerful approach for the study of low populated, invisible conformations of biological molecules. One of the powerful features of the experiment is that chemical shift differences between the exchanging conformers can be obtained, providing structural information about invisible excited states. Through the development of new labeling approaches and NMR experiments it is now possible to measure backbone 13C(alpha) and 13CO relaxation dispersion profiles in proteins without complications from 13C-13C couplings. Such measurements are presented here, along with those that probe exchange using 15N and 1HN nuclei. A key experimental design has been the choice of an exchanging system where excited-state chemical shifts were known from independent measurement. Thus it is possible to evaluate quantitatively the accuracy of chemical shift differences obtained in dispersion experiments and to establish that in general very accurate values can be obtained. The experimental work is supplemented by computations that suggest that similarly accurate shifts can be measured in many cases for systems with exchange rates and populations that fall within the range of those that can be quantified by relaxation dispersion. The accuracy of the extracted chemical shifts opens up the possibility of obtaining quantitative structural information of invisible states of the sort that is now available from chemical shifts recorded on ground states of proteins.

Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding

Many experimental and theoretical studies have suggested a significant role for nonnative interactions in protein folding pathways, but the energetic contributions of these interactions are not well understood. We have addressed the energetics and the position specificity of nonnative hydrophobic interactions by developing a continuum coarse-grained chain model with a native-centric potential augmented by sequence-dependent hydrophobic interactions. By modeling the effect of different hydrophobicity values at various positions in the Fyn SH3 domain, we predicted energetically significant nonnative interactions that led to acceleration or deceleration of the folding rate depending on whether they were more populated in the transition state or unfolded state. These nonnative contacts were centered on position 53 in the Fyn SH3 domain, which lies in an exposed position in a 310-helix. The energetic importance of the predicted nonnative interactions was confirmed experimentally by folding kinetics studies combined with double mutant thermodynamic cycles. By attaining agreement of theoretical and experimental investigations, this study provides a compelling demonstration that specific nonnative interactions can significantly influence folding energetics. Moreover, we show that a coarse-grained model with a simple consideration of hydrophobicity is sufficient for the accurate prediction of kinetically important nonnative interactions.

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.

Determination of Isoleucine Side-Chain Conformations in Ground and Excited States of Proteins from Chemical Shifts

A simple method is presented for quantifying Ile chi(2) rotamer distributions in proteins based on the measurement of Ile (13)C(delta1) chemical shifts. The methodology is well suited for applications involving very high molecular weight protein complexes, where other NMR parameters such as side-chain scalar coupling constants that report on dihedral angles cannot be measured or for studies of invisible, excited protein states, where chemical shifts are obtained from analysis of CPMG relaxation dispersion profiles. The utility of the approach is demonstrated by an application to the folding reaction of a mutant Fyn SH3 domain, where Ile side-chain structure and dynamics of an on-folding pathway intermediate state are studied.

High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2

Allergic reactions to peanuts are a serious health problem because of their high prevalence, associated with potential severity, and chronicity. One of the three major allergens in peanut, Ara h 2, is a member of the conglutin family of seed storage proteins. Ara h 2 shows high sequence homology to proteins of the 2S albumin family. Presently, only very few structural data from allergenic proteins of this family exist. For a detailed understanding of the molecular mechanisms of food-induced allergies and for the development of therapeutic strategies knowledge of the high-resolution three-dimensional structure of allergenic proteins is essential. We report a method for the efficient large-scale preparation of properly folded Ara h 2 for structural studies and report CD-spectroscopic data. In contrast to other allergenic 2S albumins, Ara h 2 exists as a single continuous polypeptide chain in peanut seeds, and thus heterologous expression in Escherichia coli was possible. Ara h 2 was expressed as Trx-His-tag fusion protein in E. coli Origami (DE3), a modified E. coli strain with oxidizing cytoplasm which allows the formation of disulfide bridges. It could be shown that recombinant Ara h 2, thus overexpressed and purified, and the allergen isolated from peanuts are identical as judged from immunoblotting, analytical HPLC, and circular dichroism spectra.

Structural Properties and Dynamic Behavior of Nonfibrillar Oligomers Formed by PrP(106−126)

The formation of nonfibrillar oligomers has been proposed as a common element of the aggregation pathway of proteins and peptides associated with neurodegenerative diseases such as Alzheimers and Creutzfeldt-Jakob disease. While fibrillar structures have long been considered indicators of diseases linked with the accumulation of amyloid plaques, it has more recently been proposed that amyloid oligomers are in fact the cytotoxic form. Here we describe the local structure and dynamics of stable oligomers formed by a peptide comprising residues 106-126 of the human prion protein (PrP). Structural constraints from solid-state NMR reveal quaternary packing interactions within the hydrophobic core, similar to those previously reported for amyloid fibrils formed by this peptide, and consistent with structural studies of oligomers formed by the Alzheimers beta-amyloid peptide. However, a hydration-dependent increase in disorder is observed for nonfibrillar oligomers of PrP(106-126). In solution NMR spectra we observe narrow (1)H and (13)C resonances corresponding to a monomer in exchange with the approximately 30 nm diameter nonfibrillar oligomers, giving additional information on the molecular structure of these species. Taken together, our data support a model in which the local structure of the oligomers contains the basic elements of amyloid fibrils, but with long-range disorder and local mobility that distinguishes these assemblies from the fibrillar form of PrP(106-126). These characteristics may provide a basis for the differing biological activities of amyloid fibrils and oligomers.

Φ-Value analysis of a three-state protein folding pathway by NMR relaxation dispersion spectroscopy

Experimental studies of protein folding frequently are consistent with two-state folding kinetics. However, recent NMR relaxation dispersion studies of several fast-folding mutants of the Fyn Src homology 3 (SH3) domain have established that folding proceeds through a low-populated on-pathway intermediate, which could not be detected with stopped-flow experiments. The dispersion experiments provide precise kinetic and thermodynamic parameters that describe the folding pathway, along with a detailed site-specific structural characterization of both the intermediate and unfolded states from the NMR chemical shifts that are extracted. Here we describe NMR relaxation dispersion Φ-value analysis of the A39V/N53P/V55L Fyn SH3 domain, where the effects of suitable point mutations on the energy landscape are quantified, providing additional insight into the structure of the folding intermediate along with per-residue structural information of both rate-limiting transition states that was not available from previous studies. In addition to the advantage of delineating the full three-state folding pathway, the use of NMR relaxation dispersion as opposed to stopped-flow kinetics to quantify Φ values facilitates their interpretation because the obtained chemical shifts monitor any potential structural changes along the folding pathway that might be introduced by mutation, a significant concern in their analysis. Φ-Value analysis of several point mutations of A39V/N53P/V55L Fyn SH3 establishes that the β3–β4-hairpin already is formed in the first transition state, whereas strand β1, which forms nonnative interactions in the intermediate, does not fully adopt its native conformation until after the final transition state. The results further support the notion that on-pathway intermediates can be stabilized by nonnative contacts.

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins.

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.