DNP-enhanced MAS NMR reveals extended conformations for the DsbA signal peptide within the ribosome exit tunnel.

Abstract

Abstract

Abstract

Abstract

Tasche gefunden: Ob sich Protein-Protein-Wechselwirkungsdomänen als Angriffsstellen für Wirkstoffe eignen, ist noch umstritten. Die 3D-Struktur eines Komplexes aus einem kleinen organischen Liganden und der AF6-PDZ-Domäne lässt die Bildung einer Bindungstasche durch den Liganden erkennen (siehe Bild). Die Verbindung kann mit einem natürlichen Peptidliganden der Domäne konkurrieren und eignet sich als Baustein für die Erzeugung selektiver PDZ-Inhibitoren.

In the groove: The “drugability” of protein–protein interaction domains is still a matter of debate. The 3D structure of a complex of a small organic ligand and the AF6 PDZ domain revealed the creation of a binding pocket by the ligand (see picture). The derived compound is able to compete with a natural peptide ligand of the domain and represents a basic building block for the generation of selective PDZ inhibitors.

Uniformly¹³C-,¹⁵N-labelled outer-membrane protein G (OmpG) from Escherichia coli was expressed for structural studies by solid-state magic-angle spinning (MAS) NMR. Inclusion bodies of the recombinant, labelled protein were purified under denaturing conditions and refolded in detergent. OmpG was reconstituted into lipid bilayers and several milligrams of two-dimensional crystals were obtained. Solid-state MAS NMR spectra showed signals with an apparent line width of 80–120 Hz (including homonuclear scalar couplings). Signal patterns for several amino acids, including threonines, prolines and serines were resolved and identified in 2D proton-driven spin-diffusion (PDSD) spectra.

Protein–protein interactions are essential in every aspect of cellular activity. Multiprotein complexes form and dissociate constantly in a specifically tuned manner, often by conserved mechanisms. Protein domains that bind proline-rich motifs (PRMs) are frequently involved in signaling events. The unique properties of proline provide a mechanism for highly discriminatory recognition without requiring high affinities. We present herein a detailed, quantitative assessment of the structural features that define the interfaces between PRM-binding domains and their target PRMs, and investigate the specificity of PRM recognition. Together with the analysis of peptide-library screens, this approach has allowed the identification of several highly conserved key interactions found in all complexes of PRM-binding domains. The inhibition of protein–protein interactions by using small-molecule agents is very challenging. Therefore, it is important to first pinpoint the critical interactions that must be considered in the design of inhibitors of PRM-binding domains.

Der Flüssig-fest-Übergang: Automatische Zuordnung von Festkörper-NMR-spektroskopischen Daten sowie schnelle und genaue Berechnung der Struktur immobilisierter Proteine mit atomarer Auflösung gelingen mit dem Programm SOLARIA (einer modifizierten Version des ARIA-Protokolls). Im Bild sind die mit SOLARIA berechneten energetisch günstigsten Strukturen der SH3-Domäne von α-Spectrin (blau) mit dem Ergebnis einer Röntgenstrukturanalyse (rot) überlagert.

Protein-Protein-Wechselwirkungen sind für alle Aspekte zellulärer Aktivität wichtig. Bildung und Dissoziation der Multiproteinkomplexe erfolgen auf spezifische Weise, oft mit ähnlichen Mechanismen. Bei der Signalübertragung sind häufig Proteindomänen beteiligt, die Prolin-reiche Motive binden (PRMs); Prolin begünstigt die spezifische Erkennung derartiger Motive, ohne dass dabei hohe Affinitäten benötigt werden. In diesem Aufsatz stellen wir eine detaillierte und quantitative Bewertung der Strukturmerkmale vor, die die Wechselwirkungen zwischen den PRM-Bindungsdomänen und den PRMs der Liganden festlegen, und untersuchen die Spezifität der PRM-Erkennung. Durch Kombination mit Ergebnissen des Screenings von Peptidbibliotheken konnten wir verschiedene hoch konservierte Wechselwirkungen identifizieren, die in allen Komplexen von PRM-Bindungsdomänen vorkommen. Das Unterdrücken von Protein-Protein-Wechselwirkungen mithilfe kleiner Moleküle ist eine große Herausforderung – daher ist es wichtig, zunächst genau die kritischen Wechselwirkungen zu identifizieren, die beim Design von Inhibitoren von PRM-Bindungsdomänen zu berücksichtigen sind.

The assignment of nonexchanging protons of a small microcrystalline protein, the α-spectrin SH3 domain (7.2 kDa, 62 residues), was achieved by means of three-dimensional (3D) heteronuclear (¹H–¹³C–¹³C) magic-angle spinning (MAS) NMR dipolar correlation spectroscopy. With the favorable combination of a high B₀-field, a moderately high spinning frequency, and frequency-switched Lee-Goldburg irradiation applied during ¹H evolution, a proton linewidth ≤0.5 ppm at 17.6 Tesla was achieved for the particular protein preparation used. A comparison of the solid-state ¹H chemical shifts with the shifts found in solution shows a remarkable similarity, which reflects the identical protein structures in solution and in the solid. Significant differences between the MAS solid- and liquid-state ¹H chemical shifts are only observed for residues that are located at the surface of the protein and that exhibit contacts between different SH3 molecules. In two cases, aromatic residues of neighboring SH3 molecules induce pronounced upfield ring-current shifts for protons in the contact area.

The backbone and side-chain ¹³C and ¹⁵N signals of a solid 62-residue (u-¹³C,¹⁵N)-labelled protein containing the α-spectrin SH3 domain were assigned by two-dimensional (2D) magic angle spinning (MAS) ¹⁵N–¹³C and ¹³C–¹³C dipolar correlation spectroscopy at 17.6 T. The side-chain signal sets of the individual amino acids were identified by 2D ¹³C–¹³C proton-driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton–nitrogen and a nitrogen–carbon cross-polarisation step followed by a carbon–carbon homonuclear transfer unit. Interresidue correlations leading to sequence-specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7–61, while the signals of the N- and C-terminal residues 1–6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor-bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three-dimensional versions of the applied or analogous techniques.