Structural model of dodecameric heat-shock protein Hsp21:Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations

Studies of micelle-bound peptides using paramagnetic relaxation enhancement

Viele Peptide, Proteine Natur- und Wirkstoffe binden an biologische Membranen. Um deren Funktion auf molekularer Ebene zu verstehen ist es notwendig deren Topologie in der Membran zu kennen. In dieser Dissertation werden neue Lösungs-NMR Methodiken präsentiert um die Orientierung und Lokalisierung von helikalen Peptiden in einem Membranmimetikum zu bestimmen. Durch die Messung von Protonen T1 Relaxationsraten während einer Titration mit einer paramagnetischen Sonde erhält man ein wellenförmiges Muster mit einer Periodizität von 3.6 Aminosäurenresten pro Welle. Ich benutzte hierzu die wasserlösliche paramagnetische Substanz Gd(DTPA-BMA) da diese keine Wechselwirkungen mit Proteinen zeigt. Die Relaxationsraten bei unterschiedlichen Konzentrationen der paramagnetischen Substanz wurden aus Kreuzsignalen von saturation-recovery 2D NOESY und 2D TOCSY Spektren erhalten. Untersucht wurden das 15 Aminosäuren antimikrobielle Peptid CM15 und das aus 25 Aminosäuren bestehende Peptid TM7, welches die transmembran Helix 7 der V-ATPase aus Hefe darstellt. Beide Peptide wurden in unmarkierter Form untersucht. Aus dem erhaltenen Wellenmuster der paramagnetischen Relaxation können der Rotationswinkel (Azimuth) und der Drehwinkel (Tilt) ermittelt werden. Die Lokalisierung innerhalb der Mizelle (Eindringtiefe) wurde mittels des ParaPos Verfahrens erhalten. Dabei werden experimentelle paramagnetische Relaxationsratenerhöhungen (PREs) von Protonen korreliert mit deren Eindringtiefe nach Kalibration an einem System bekannter Topologie - im speziellen Fall der transmembran Helix TM7. Die Orientierung und Eindringtiefe von TM7 und CM15 wurden durch least-square fitting der experimentellen PREs berechnet. Die präsentierte Methode ermöglicht die Bestimmung der Orientierung und Lokalisierung eines Peptides in einer Mizelle auch an unmarkierten Molekülen ohne kovalente Modifikation des Peptides und eröffnet die Möglichkeit die Topologie auch anderer mizell-gebundener Substanzen zu ermitteln.Many peptides, proteins, natural compounds and drugs bind to biological membranes. Determining their topology is crucial in understanding their function and activity on a molecular level. Here new solution state NMR methods for obtaining the complete orientation and location of helical peptides in a membrane-mimetic environment (micelle-bound) are presented. By monitoring proton T1 - relaxation rates during a titration with a paramagnetic surface probe, a wave-like pattern with a periodicity of 3.6 residues per turn was obtained. The water-soluble paramagnetic agent Gd(DTPA-BMA) was used due to its reported absence of interactions with proteins. The relaxation rates at different concentrations of the paramagnetic compound were derived from the cross-peaks of saturation-recovery 2D-NOESY and 2D-TOCSY spectra on the antimicrobial fifteen residues peptide CM15 and the twenty five residues peptide TM7 that mimics the Vph1p transmembrane helix 7 of yeast V-ATPase, both in unlabeled form. The obtained wave-like pattern, the paramagnetic relaxation wave, allows the determination of the rotation (azimuth) and tilt angle of the helix. The location of the peptides in terms of their immersion depths was obtained based on the ParaPos approach. The measured paramagnetic relaxation enhancements (PREs) of protons were correlated with their micellar immersion depths after calibration using a system of known topology, in this case a transmembrane helical peptide TM7. The orientation and immersion depth of the TM7 and CM15 peptides were then obtained by least-square fitting of measured versus calculated PREs.The presented methods enable the complete orientation and location of the peptide in the micelle to be obtained even on unlabeled peptides by one simple experiment that does not require any covalent modifications of the peptide. The approaches also open a path towards the topology determination of any structurally characterized micelle bound molecule.Michal RespondekAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersGraz, Univ., Diss., 2008OeBB(VLID)20118

Conformational exchange of aromatic side chains characterized by L-optimized TROSY-selected (13)C CPMG relaxation dispersion.

Protein dynamics on the millisecond time scale commonly reflect conformational transitions between distinct functional states. NMR relaxation dispersion experiments have provided important insights into biologically relevant dynamics with site-specific resolution, primarily targeting the protein backbone and methyl-bearing side chains. Aromatic side chains represent attractive probes of protein dynamics because they are over-represented in protein binding interfaces, play critical roles in enzyme catalysis, and form an important part of the core. Here we introduce a method to characterize millisecond conformational exchange of aromatic side chains in selectively (13)C labeled proteins by means of longitudinal- and transverse-relaxation optimized CPMG relaxation dispersion. By monitoring (13)C relaxation in a spin-state selective manner, significant sensitivity enhancement can be achieved in terms of both signal intensity and the relative exchange contribution to transverse relaxation. Further signal enhancement results from optimizing the longitudinal relaxation recovery of the covalently attached (1)H spins. We validated the L-TROSY-CPMG experiment by measuring fast folding-unfolding kinetics of the small protein CspB under native conditions. The determined unfolding rate matches perfectly with previous results from stopped-flow kinetics. The CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained by urea-dependent chemical shift analysis. The present method enables characterization of conformational exchange involving aromatic side chains and should serve as a valuable complement to methods developed for other types of protein side chains

Slow Aromatic Ring Flips Detected Despite Near-Degenerate NMR Frequencies of the Exchanging Nuclei.

Aromatic ring flips of Phe and Tyr residues are a hallmark of protein dynamics with a long history in molecular biophysics. Ring flips lead to symmetric exchange of nuclei between sites with distinct magnetic environments, which can be probed by NMR spectroscopy. Current knowledge of ring-flip rates originates from rare cases in which the chemical shift difference between the two sites is sufficiently large and the ring-flip rate sufficiently slow, typically kflip < 10(3) s(-1), so that separate peaks are observed in the NMR spectrum for the two nuclei, enabling direct measurement of the flip rate. By contrast, a great majority of aromatic rings show single peaks for each of the pairs of δ or ε nuclei, which commonly are taken as inferential evidence that the flip rate is fast, kflip ≫ 10(3) s(-1), even though rate measurements have not been achieved. Here we report a novel approach that makes it possible to identify slow ring flips in previously inaccessible cases where only single peaks are observed. We demonstrate that Y21 in the bovine pancreatic trypsin inhibitor (BPTI) has a slow ring-flip rate, kflip < 100 s(-1), a result that contrasts with previous estimates of 10(4)-10(6) s(-1) inferred from the single-peak spectrum of Y21. Comparison with a recent 1 ms molecular dynamics trajectory of BPTI shows qualitative agreement and highlights the value of accurate aromatic ring flip data as an important benchmark for molecular dynamics simulations of proteins across wide time scales

Cu/Zn Superoxide Dismutase Forms Amyloid Fibrils under Near-Physiological Quiescent Conditions : The Roles of Disulfide Bonds and Effects of Denaturant

Cu/Zn superoxide dismutase (SOD1) forms intracellular aggregates that are pathological indicators of amyotrophic lateral sclerosis. A large body of research indicates that the entry point to aggregate formation is a monomeric, metal-ion free (apo), and disulfide-reduced species. Fibril formation by SOD1 in vitro has typically been reported only for harsh solvent conditions or mechanical agitation. Here we show that monomeric apo-SOD1 in the disulfide-reduced state forms fibrillar aggregates under near-physiological quiescent conditions. Monomeric apo-SOD1 with an intact intramolecular disulfide bond is highly resistant to aggregation under the same conditions. A cysteine-free variant of SOD1 exhibits fibrillization behavior and fibril morphology identical to those of disulfide-reduced SOD1, firmly establishing that intermolecular disulfide bonds or intramolecular disulfide shuffling are not required for aggregation and fibril formation. The decreased lag time for fibril formation resulting from reduction of the intramolecular disulfide bond thus primarily reflects the decreased stability of the folded state relative to partially unfolded states, rather than an active role of free sulfhydryl groups in mediating aggregation. Addition of urea to increase the amount of fully unfolded SOD1 increases the lag time for fibril formation, indicating that the population of this species does not dominate over other factors in determining the onset of aggregation. Our results contrast with previous results obtained for agitated samples, in which case amyloid formation was accelerated by denaturant. We reconcile these observations by suggesting that denaturants destabilize monomeric and aggregated species to different extents and thus affect nucleation and growth

Slow Aromatic Ring Flips Detected Despite Near-Degenerate NMR Frequencies of the Exchanging Nuclei

Aromatic ring flips of Phe and Tyr residues are a hallmark of protein dynamics with a long history in molecular biophysics. Ring flips lead to symmetric exchange of nuclei between sites with distinct magnetic environments, which can be probed by NMR spectroscopy. Current knowledge of ring-flip rates originates from rare cases in which the chemical shift difference between the two sites is sufficiently large and the ring-flip rate sufficiently slow, typically <i>k</i>_flip < 10³ s^–1, so that separate peaks are observed in the NMR spectrum for the two nuclei, enabling direct measurement of the flip rate. By contrast, a great majority of aromatic rings show single peaks for each of the pairs of δ or ε nuclei, which commonly are taken as inferential evidence that the flip rate is fast, <i>k</i>_flip ≫ 10³ s^–1, even though rate measurements have not been achieved. Here we report a novel approach that makes it possible to identify slow ring flips in previously inaccessible cases where only single peaks are observed. We demonstrate that Y21 in the bovine pancreatic trypsin inhibitor (BPTI) has a slow ring-flip rate, <i>k</i>_flip < 100 s^–1, a result that contrasts with previous estimates of 10⁴–10⁶ s^–1 inferred from the single-peak spectrum of Y21. Comparison with a recent 1 ms molecular dynamics trajectory of BPTI shows qualitative agreement and highlights the value of accurate aromatic ring flip data as an important benchmark for molecular dynamics simulations of proteins across wide time scales

Structural basis for nucleic acid and toxin recognition of the bacterial antitoxin CcdA.

Toxin-antitoxin systems are highly abundant in plasmids and bacterial chromosomes. They ensure plasmid maintenance by killing bacteria that have lost the plasmid. Their expression is autoregulated at the level of transcription. Here, we present the solution structure of CcdA, the antitoxin of the ccd system, as a free protein (16.7 kDa) and in complex with its cognate DNA (25.3 kDa). CcdA is composed of two distinct and independent domains: the N-terminal domain, responsible for DNA binding, which establishes a new family of the ribbon-helix-helix fold and the C-terminal region, which is responsible for the interaction with the toxin CcdB. The C-terminal domain is intrinsically unstructured and forms a tight complex with the toxin. We show that CcdA specifically recognizes a 6 bp palindromic DNA sequence within the operator-promoter (OP) region of the ccd operon and binds to DNA by insertion of the positively charged N-terminal beta-sheet into the major groove. The binding of up to three CcdA dimers to a 33mer DNA of its operator-promoter region was studied by NMR spectroscopy, isothermal titration calorimetry and single point mutation. The highly flexible C-terminal region of free CcdA explains its susceptibility to proteolysis by the Lon ATP-dependent protease.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Structural and Thermodynamic Characterization of Vibrio fischeri CcdB*

CcdBVfi from Vibrio fischeri is a member of the CcdB family of toxins that poison covalent gyrase-DNA complexes. In solution CcdBVfi is a dimer that unfolds to the corresponding monomeric components in a two-state fashion. In the unfolded state, the monomer retains a partial secondary structure. This observation correlates well with the crystal and NMR structures of the protein, which show a dimer with a hydrophobic core crossing the dimer interface. In contrast to its F plasmid homologue, CcdBVfi possesses a rigid dimer interface, and the apparent relative rotations of the two subunits are due to structural plasticity of the monomer. CcdBVfi shows a number of non-conservative substitutions compared with the F plasmid protein in both the CcdA and the gyrase binding sites. Although variation in the CcdA interaction site likely determines toxin-antitoxin specificity, substitutions in the gyrase-interacting region may have more profound functional implications