Solid-state NMR study of the YadA membrane-anchor domain in the bacterial outer membrane.

MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using 13C-13C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments

Festkörper-NMR-Studien an der Membrananker-Domäne von YadA in der bakteriellen Außenmembran.

MAS-NMR-Spektroskopie wurde verwendet, um die Struktur und Dynamik der Membrananker-Domäne von YadA (YadA-M) direkt in der bakteriellen Außenmembran zu untersuchen, in der das Protein exprimiert wurde. YadA ist ein Adhäsin des Pathogens Yersinia enterocolitica, das an Interaktionen mit Wirtszellen beteiligt ist und darüber hinaus auch als Modellprotein zu Studien des Autotransportprozesses dient. Mithilfe von 13C-13C-DARR-und PDSD-Spektren bei unterschiedlichen Mischzeiten konnten wir existierende chemische Verschiebungen für einen weiten Bereich von YadA-M in der Lipidmembran übertragen. Die chemischen Verschiebungen von YadA-M waren in den meisten Regionen im Vergleich zu denen in mikrokristallinen Präparationen, auf deren Basis bereits eine Struktur gelöst wurde, unverändert. Das trifft insbesondere auf die ASSA-Region zu, die vermutlich als Übergangszustand während des Transports der löslichen Domäne eine Haarnadelstruktur einnimmt. Vergleiche der Dynamik zwischen mikrokristallinen und in der Membran eingebetteten Proben zeigen eine höhere Flexibilität der ASSA-Region in den Außenmembran-Präparationen bei physiologischen Temperaturen. Diese Studie wird den Weg für die MAS-NMR-basierte Strukturberechnung von Membranproteinen und für ein besseres Verständnis der Funktion von dynamischen Proteinbereichen in nativen Membranen bereiten

Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with beta-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical beta-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents

Robust, Integrated Computational Control of NMR Experiments to Achieve Optimal Assignment by ADAPT-NMR

ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) represents a groundbreaking prototype for automated protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. With a [13C,15N]-labeled protein sample loaded into the NMR spectrometer, ADAPT-NMR delivers complete backbone resonance assignments and secondary structure in an optimal fashion without human intervention. ADAPT-NMR achieves this by implementing a strategy in which the goal of optimal assignment in each step determines the subsequent step by analyzing the current sum of available data. ADAPT-NMR is the first iterative and fully automated approach designed specifically for the optimal assignment of proteins with fast data collection as a byproduct of this goal. ADAPT-NMR evaluates the current spectral information, and uses a goal-directed objective function to select the optimal next data collection step(s) and then directs the NMR spectrometer to collect the selected data set. ADAPT-NMR extracts peak positions from the newly collected data and uses this information in updating the analysis resonance assignments and secondary structure. The goal-directed objective function then defines the next data collection step. The procedure continues until the collected data support comprehensive peak identification, resonance assignments at the desired level of completeness, and protein secondary structure. We present test cases in which ADAPT-NMR achieved results in two days or less that would have taken two months or more by manual approaches

A unified model for BAM function that takes into account type Vc secretion and species differences in BAM composition

Transmembrane proteins in the outer membrane of Gram-negative bacteria are almost exclusively β-barrels. They are inserted into the outer membrane by a conserved and essential protein complex called the BAM (for β-barrel assembly machinery). In this commentary, we summarize current research into the mechanism of this protein complex and how it relates to type V secretion. Type V secretion systems are autotransporters that all contain a β-barrel transmembrane domain inserted by BAM. In type Vc systems, this domain is a homotrimer. We argue that none of the current models are sufficient to explain BAM function particularly regarding type Vc secretion. We also find that current models based on the well-studied model system Escherichia coli mostly ignore the pronounced differences in BAM composition between different bacterial species. We propose a more holistic view on how all OMPs, including autotransporters, are incorporated into the lipid bilayer

Influence of different assignment conditions on the determination of symmetric homodimeric structures with ARIA

The ambiguous restraint for iterative assignment (ARIA) approach for NMR structure calculation is evaluated for symmetric homodimeric proteins by assessing the effect of several data analysis and assignment methods on the structure quality. In particular, we study the effects of network anchoring and spin-diffusion correction. The spin-diffusion correction improves the protein structure quality systematically, whereas network anchoring enhances the assignment efficiency by speeding up the convergence and coping with highly ambiguous data. For some homodimeric folds, network anchoring has been proved essential for unraveling both chain and proton assignment ambiguities

ARIA2: automated NOE assignment and data integration in NMR structure calculation

Modern structural genomics projects demand for integrated methods for the interpretation and storage of nuclear magnetic resonance (NMR) data. Here we present version 2.1 of our program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of nuclear Overhauser enhancement (NOE) data and NMR structure calculation. We report on recent developments, most notably a graphical user interface, and the incorporation of the object-oriented data model of the Collaborative Computing Project for NMR (CCPN). The CCPN data model defines a storage model for NMR data, which greatly facilitates the transfer of data between different NMR software packages