Solution Structure of Human Proguanylin: The role of a hormone prosequence

The endogenous ligand of guanylyl cyclase C, guanylin, is produced as the 94-amino-acid prohormone proguanylin, with the hormone guanylin located at the COOH terminus of the prohormone. The solution structure of proguanylin adopts a new protein fold and consists of a three-helix bundle, a small three-stranded {beta}-sheet of two NH2-terminal strands and one COOH-terminal strand, and an unstructured linker region. The sequence corresponding to guanylin is fixed in its bioactive topology and is involved in interactions with the NH2-terminal {beta}-hairpin: the hormone region (residues 80–94) partly wraps around the first 4 NH2-terminal residues that thereby shield parts of the hormone surface. These interactions provide an explanation for the negligible bioactivity of the prohormone as well as the important role of the NH2-terminal residues in the disulfide-coupled folding of proguanylin. Since the ligand binding region of guanylyl cyclase C is predicted to be located around an exposed {beta}-strand, the intramolecular interactions observed between guanylin and its prosequence may be comparable with the guanylin/receptor interaction

Protein Structure, Dynamics and Interactions at Atomic Resolution from Solution NMR Spectroscopy

Pioneering work in the 1980s has established NMR spectroscopy as a routine method for protein structure determination with spatial resolution rivaling X-ray crystallography, and recent technological and methodological advances have significantly widened the scope and practical applicability of protein NMR. Understanding protein folding and function obviously requires knowledge not only of static structures but also of the conformational dynamics. Because the spectral parameters are sensitive to dynamics on all time-scales from picoseconds to real time, NMR spectroscopy is a particularly powerful tool for studying protein folding, intrinsically disordered regions, protein-protein and protein-ligand interactions, enzyme catalysis and other dynamic processes under native solution conditions, uniquely combining high spatial and temporal resolution. Following an introduction into the principles of NMR spectroscopy and an overview over various techniques in the state-of-the-art protein NMR “toolbox” with their advantages and limitations, the elucidation of the folding pathway of the Fyn SH3 A39V/N53P/V55L domain at high spatial and temporal resolution is presented as an example. This domain folds into its native conformation via a low-populated transient intermediate with a lifetime in the millisecond range. Folding intermediates have long been implicated in amyloid fibril formation involved in neurodegenerative disorders but the structural mechanisms have remained largely elusive. The intermediate of the Fyn SH3 A39V/N53P/V55L exposes an aggregation-prone beta-strand and mutants mimicking the intermediate spontaneously form fibrillar aggregates. This study provides detailed insight into how non-native interactions can derail folding and initiate amyloid fibril formation

Purification and Characterization of Recombinant N-Terminally Pyroglutamate-Modified Amyloid-β Variants and Structural Analysis by Solution NMR Spectroscopy

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEAβ from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-15N]- or [U-13C, 15N]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified Aβ peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEAβ40 and pEAβ42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEAβ40 and pEAβ42 for further physiological and biochemical studies

Solution NMR study of the interaction of HIV-1 VpU and human BST-2

The human protein BST-2, also known as tetherin/CD317/HM1.24, is an important component of antiviral defence that restricts enveloped viruses from budding from the host cell [1]. Virus protein “U” (VpU), an accessory protein of human immunodeficiency virus type 1 (HIV-1), is able to antagonize and downregulate BST-2 in infected cells [2]. The presence of VpU increases HIV-1 replication by one order of magnitude [3].We have studied the interaction of the two bitopic transmembrane (TM) proteins BST-2 and VpU in membrane mimicking DPC micelles using solution state nuclear magnetic resonance (NMR) spectroscopy. An E. coli-based cell-free expression (CFE) system was used for the production of isotope-labelled proteins, i.e., full-length VpU and a BST-2 construct that encompasses the cytoplasmic and the single transmembrane domain of this protein. Resonance assignment of backbone nuclei of the BST-2 construct is close to complete and allowed secondary structure analysis based on secondary chemical shifts. In particular, an α-helical stretch of amino acids was confirmed in the predicted TM region of BST-2. Further, isotope-labelled BST-2 was titrated with increasing amounts of unlabelled VpU in order to identify BST-2 residues that intimately approach VpU in the putative BST-2/VpU complex in DPC micelles. The observed chemical shift changes map to the TM helix of BST-2. Resonance assignment of full-length VpU is currently in progress and will enable a more detailed analysis of the BST-2 VpU complex. [1] Hammonds J., et al, PLoS Pathog. 6(2): e1000749 (2010) [2] Dubé M., et al, PLoS Pathog. 6(4): e1000856 (2010)[3] Fischer W.B., FEBS Lett. 552:39-46 (2003

Multiple WW domains of Nedd4-1 undergo conformational exchange that is quenched upon peptide binding

The third WW domain (WW3*) of the ubiquitin ligase human neuronal precursor cell expressed developmentally downregulated gene 4-1 (hNedd4-1) was reported to bind its PY motif peptide by a coupled folding-binding equilibrium. However, it is unknown whether these thermodynamic properties are retained in the context of neighboring hNedd4-1 domains. In this report, NMR data show that the WW3* displays a fold-unfold equilibrium in the presence of neighboring WW domains, and that similar fold-unfold equilibria also likely exist for neighboring WW domains. These equilibria are quenched upon interaction with peptide. Thus, the binding mechanism of hNedd4-1 WW domains to proteins involves coupled folding and binding equilibria, and this mechanism may be a general feature that modulates peptide affinities of WW domains

Conformational dynamics of the autophagy-related protein GABARAP on multiple time-scales

Understanding the function of a protein usually requires knowledge of its tertiary structure and conformationaldynamics. NMR spectroscopy is a powerful tool for studying structure and dynamics on virtually all time-scales frompicoseconds to real time at atomic resolution. In particular, sub-nanosecond dynamics determine spin relaxationrates, whereas the biochemically often more relevant dynamics on the micro- to millisecond time-scale causes linebroadening, which can be quantified by Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion (RD) experiments[1].The 117-residue GABAA receptor-associated protein (GABARAP) from H. sapiens is known to mediate vesicletransport and fusion events in autophagy and possibly also apoptosis [2]. To this end, GABARAP is enzymaticallylipid-conjugated to allow membrane anchoring, which has been reported to facilitate hemifusion of membranes uponoligomerization. Structure determination of GABARAP by NMR [3] and X-ray crystallography [4] suggested significantconformational heterogeneity, which is conserved in the yeast homologue Atg8 [2]. Intriguingly, crystallizationunder high salt conditions resulted in an alternate conformation in which the N-terminal region is associated withthe hydrophobic binding pockets of an adjacent subunit in the crystal [4]. Unfortunately, it remains unclear whetherthis alternate conformation indeed facilitates oligomerization during membrane fusion and/or tubulin polymerizationor is merely a crystallization artifact, and the conformational dynamics of GABARAP is still poorly understood.To gain insight, we have measured 15N longitudinal and transverse relaxation rates, {1H}15N heteronuclear NOEs,and 15N relaxation dispersion profiles at several different temperatures, which reveal conformational dynamics invarious regions of the tertiary structure. The C-terminal region is highly mobile on the nanosecond time-scale asindicated by low order parameters S2. CPMG RD experiments show two distinct conformational exchange processeson the millisecond time-scale. Specifically, resonances in the N-terminal helical subdomain exhibit two separate resonances(plus exchange cross-peaks in multidimensional experiments) as a result of slow to intermediate exchangeon a time-scale of several milliseconds between two conformations with similar equilibrium populations. By contrast,residues lining the hydrophobic binding pockets reveal a slightly faster exchange process with an excited statepopulation of about 1%. Investigation of the structural details of these conformational exchange processes via CPMGRD experiments on a variety of different nuclei is currently underway.References: [1] A. G. Palmer 2004, Chem. Rev. 104, 3623-3640. [2] M. Schwarten et al. 2010, Biochem. Biophys. Res. Commun.395, 426-431. [3] T. Stangler et al. 2002, J. Biol. Chem. 277, 13363-13366. [4] J. E. Coyle et al. 2002, Neuron 33, 63-74.(RD 717, Poster S. 334 von 438

High-Affinity Binding of Monomeric but Not Oligomeric Amyloid-β to Ganglioside GM1 Containing Nanodiscs

The interaction of the amyloid-β protein (Aβ) with neuronal cell membranes plays a crucial role in Alzheimer’s disease. Aβ undergoes structural changes upon binding to ganglioside GM1 containing membranes leading to altered molecular characteristics of the protein. The physiological role of the Aβ interaction with the ganglioside GM1 is still unclear. In order to further elucidate the molecular requirements of Aβ membrane binding, we tested different nanodiscs varying in their lipid composition, regarding the charge of the headgroups as well as ganglioside GM1 concentration. Nanodiscs are excellent model membrane systems for studying protein membrane interactions, and we show here their suitability to investigate the membrane interaction of Aβ. In particular, we set out to investigate whether the binding activity of GM1 to Aβ is specific for the assembly state of Aβ and compared the binding affinities of monomeric with oligomeric Aβ. Using fluorescence titration experiments, we demonstrate high-affinity binding of Aβ(1−40) to GM1 containing nanodiscs, with dissociation constants, KD, in the range from 25 to 41 nM, in a GM1 concentration-dependent manner. Biolayer interferometry experiments confirmed the high-affinity binding of monomeric Aβ(1−40) (KD of 24 nM to 49 nM) as well as of Aβ(1−42) (KD of 30 nM) to GM1 containing nanodiscs, and no binding to phospholipid containing nanodiscs. Interestingly, and in contrast to monomeric Aβ, neither oligomeric Aβ(1−40) nor oligomeric Aβ(1−42) binds to GM1 nanodiscs. To the best of our knowledge, this is the first report of a loss of function for monomeric Aβ upon aggregation