The crystal structure of the C-terminal domain of the salmonella enterica pduo protein: An old fold with a new heme-binding mode

The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 \uc5 resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (Kd = 1.6 7 10 127 M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family

Conformational heterogeneity of the Roc domains in C. tepidum Roc-COR and implications for human LRRK2 Parkinson mutations

Ras of complex proteins (Roc) is a Ras-like GTP binding domain that always occurs in tandem with the C-terminal of Roc (COR) domain, and is found in bacteria, plants and animals. Recently, it has been shown that Roco proteins belong to the family of G-proteins activated by nucleotide-dependent dimerization (GADs). We investigated the RocCOR tandem from the bacteria Chlorobium tepidum with site-directed spin labeling and pulse EPR distance measurements to follow conformational changes during the Roco G-protein cycle. Our results confirm that the COR domains are a stable dimerization device serving as a scaffold for the Roc domains, that in contrast are structurally heterogeneous and dynamic entities. Contrary to other GAD proteins, we observed only minor structural alterations upon binding and hydrolysis of GTP, indicating significant mechanistic variations within this protein class. Mutations in the most prominent member of the Roco family of proteins, leucine-rich repeat kinase 2 (LRRK2), are the most frequent cause of late-onset Parkinson's disease (PD). Using a stable recombinant LRRK2 Roc-COR-Kinase fragment we obtained detailed kinetic data for the G-protein cycle. Our data confirmed that dimerization is essential for efficient GTP hydrolysis, and PD mutations in the Roc domain result in decreased GTPase activity. Previous data have shown that these LRRK2 PD-mutations are located in the interface between Roc and COR. Importantly, analogous mutations in the conserved C. tepidum RocCOR interface significantly influence the structure and nucleotide-induced conformational changes of the Roc domains

Characterization of multifunctional β-NaEuF4/NaGdF4 core–shell nanoparticles with narrow size distribution

We characterized NaEuF4/NaGdF4 core–shell nanoparticles

Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response

The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations

Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy

The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD

Simulation vs. Reality: A Comparison of In Silico Distance Predictions with DEER and FRET Measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions

Strukturelle und funktionelle Untersuchungen des photophoben Rezeptor/Transducer-Komplexes aus Natronobacterium pharaonis

Das sensorische Rhodopsin II aus dem Archaebakterium Natronobakterium pharaonis (NpSRII) ist verantwortlich für die photophobe Reaktion des Bakteriums auf blaues und grünes Licht. Dieser Photorezeptor bildet in der Membran einen 2:2-Komplex mit einem anderen Membranprotein, dem Transducer NpHtrII, der grosse strukturelle und funktionelle Homologien zu den eubakteriellen Chemorezeptoren aufweist. Nach Lichtanregung übeträgt das NpSRII ein Signal auf das NpHtrII, welches diese Information an die gekoppelte Signaltransduktionskaskade im Inneren der Zelle weiterleitet. Die diesem Prozess zugrundeliegenden Mechanismen wurden mit den Methoden der Lichtblitz-Induzierten zeitaufgelösten Absorptionsspektroskopie, der ESR-Spektroskopie von spinmarkierten Proteinen sowie der Röntgenkristallstrukturanalyse untersucht. Mittels zeitaufgelöster ESR-Spektroskopie konnte die Kinetik der Bildung des aktivierten Rezeptorzustandes ermittelt werden. Diese korreliert mit einem Übergang im sogenannten Photozyklus des Rezeptormoleküls, welcher nicht mit einer Transprotonierung innerhalb des Proteins gekoppelt ist, wie bisher angenommen. Auf der Grundlage von Strukturinformationen, welche ESR-Spektroskopisch gewonnen wurden, konnte zudem ein Modell des transmembranen Bereiches des Rezeptor/Transducer-Komplexes erstellt werden, welches weitere Aufschlüsse bezüglich des Mechanismus der Signalübertragung zwischen den beiden Proteinen erlaubte. Zudem gelang es, diesen Bereich des Komplexes auch für die Röntgenstrukturanalyse zugänglich zu machen. Mittels der Methode der Kristallisation in der sogenannten kubischen Lipidphase gelang es damit erstmals die Kristall-Struktur eines Komplexes von Membran-Proteinen aufzuklären. Durch die so gewonnenen Struktur-Daten konnte das Modell des Signalübertragungsmechanismus verfeinert werden. Anhand der vorliegenden Ergebnisse wird davon ausgegangen, dass die bereits zuvor postulierte Ausklappbewegung der Rezeptor-Helix F eine Schraubenbewegung der zweiten Transducer-Helix induziert. Im, sich dieser Transducer-Helix anschliessenden, Linker-Bereich wurde eine parallel zur Zellmembran verlaufende Helix identifiziert, welche über einen denkbaren Hebelarmmechanismus zu einer Verstärkung des Signals führt.Sensory Rhodopsin II from the archaebacterium Natronobacterium pharaonis (NpSRII) is responsible for the photophobic response of the bacterium to blue and green light. This photoreceptor forms a 2:2-complex in the membrane with another membrane-protein, the transducer NpHtrII, which shows strong functional and structural homologies to the eubacterial chemoreceptors. After light induction a signal is transferred from NpSRII to NpHtrII, which transmits this information to the coupled signal-transduction cascade. The mechanisms underlying this process were studied using the methods of laserflash photolysis, EPR-spectroscopy on spin-labeled proteins and X-Ray-Crystallography. Applying the method of time-resolved EPR-measurements the kinetics of the formation of the active receptor state were resolved. The timecourse of this process correlates with a specific transition in the so called photocycle of the receptor molecule, which is not coupled to a transprotonation in the molecule as it was postulated up to date. On the basis of structural data obtained by EPR-spectroscopy a model for the transmembrane part of the receptor/transducer complex was developed, leading to a more refined model for the mechanism of signal transfer between the molecules. Additionally the transmembrane part of this complex was successfully crystallized in the so called lipidic cubic phase and the structure was solved by X-ray analysis. Herewith the X-ray structure of a complex of membrane proteins was resolved for the first time. The obtained structural data was used for further refinement of the model for the signalling mechanism. It was postulated on the basis of this data, that by the recently shown outward tilt of the receptor helix F a screw-like motion of the second transducer helix is induced. An additional Helix succeeding this transducer-helix, oriented parallel to the membrane, was identified, leading to a mechanism in which it acts as a lever-arm to amplify the observed signal