Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

Background: The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings: We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the b-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance: Our structural analysis provides a new insight into the mechanism by which MotB inserts into th

Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine

Dihydrodipicolinate synthase (DHDPS) catalyzes the rate limiting step in lysine biosynthesis in bacteria and plants. The structure of DHDPS has been determined from several bacterial species and shown in most cases to form a homotetramer or dimer of dimers. However, only one plant DHDPS structure has been determined to date from the wild tobacco species, Nicotiana sylvestris (Blickling et al. (1997) J. Mol. Biol. 274, 608–621). Whilst N. sylvestris DHDPS also forms a homotetramer, the plant enzyme adopts a ‘back-to-back’ dimer of dimers compared to the ‘head-to-head’ architecture observed for bacterial DHDPS tetramers. This raises the question of whether the alternative quaternary architecture observed for N. sylvestris DHDPS is common to all plant DHDPS enzymes. Here, we describe the structure of DHDPS from the grapevine plant, Vitis vinifera, and show using analytical ultracentrifugation, small-angle X-ray scattering and X-ray crystallography that V. vinifera DHDPS forms a ‘back-to-back’ homotetramer, consistent with N. sylvestris DHDPS. This study is the first to demonstrate using both crystal and solution state measurements that DHDPS from the grapevine plant adopts an alternative tetrameric architecture to the bacterial form, which is important for optimizing protein dynamics as suggested by molecular dynamics simulations reported in this study

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Herbst / multibracket appliance treatment in adult Class II:1 malocclusions

Das Ziel der vorliegenden Arbeit war es, das Ausmaß der dentoskelettalen und fazialen Veränderungen bei der Distalbissbehandlung (Klasse II-1) von Erwachsenen mit einer Herbst- /Multibracket-Apparatur anhand der Auswertung von Fernröntgenseitenbildern des Kopfes (FRS) zu ermitteln. Das Patientengut umfasste 23 erwachsene Probanden (4 männliche und 19 weibliche) mit einer Angle-Klasse-II-1. Das Alter der Probanden lag am Anfang der Behandlung durchschnittlich bei 21,9 Jahren. Alle Probanden wurden mit einer Herbst- und anschließender Multibracket-Apparatur behandelt. Fernröntgenseitenbilder (FRS) des Kopfes der Probanden wurden zu drei Zeitpunkten analysiert: T1: vor der Herbst-Behandlung T2: nach der Herbst-Behandlung T3: nach der anschließenden Multibracket-Behandlung Die Veränderungen der sagittalen Okklusion, der sagittalen und vertikalen Kieferrelation sowie der Gesichtshöhe, Profilkonvexität und der Lippenposition wurden ermittelt. Die kephalometrischen Veränderungen während drei Untersuchungs-zeiträumen wurden festgehalten: T2-T1: Herbst-Phase T3-T2: Multibracket-Phase (MB-Phase) T3-T1: Totaler Behandlungszeitraum Die Untersuchung lieferte folgende Ergebnisse: Der Overjet wurde während der Herbst-Phase um 9,98 mm korrigiert. Die Veränderungen waren zu 79 % dental und 21 % skelettal bedingt. Die Molarenrelation verbesserte sich um 6,82 mm. Dies war zu 69 % dental und 31 % skelettal bedingt. In der nachfolgenden MB-Phase rezidivierte der Overjet und die Molarenrelation leicht. Während des totalen Behandlungszeitraumes kam es in allen Fällen zu einer Korrektur des Distalbisses (Normalisierung des Overjets und der sagittalen Molarenrelation). Die Overjet-Korrektur von 6,75 mm teilte sich in 87 % dentale und 13 % skelettale Veränderungen. Die Korrektur der Molarenrelation von 4,11 mm bestand aus 78 % dentalen und 22 % skelettalen Veränderungen. Die sagittale Kieferrelation wurde während der Herbst-Phase signifikant (p<0,001) verändert. Vergrößert wurden die Winkel SNB (1,22°) und SNPg (0,93°), verkleinert wurden die Winkel ANB (1,22°) und ANPg (0,95°) sowie der Wits-Wert (2,30 mm). In der MB-Phase veränderten sich die Werte wieder leicht rückläufig. Während des totalen Behandlungszeitraumes kam es zu einer signifikanten (p<0,001) Vergrößerung der Winkel SNB (0,82°) und SNPg (0,70°) sowie zu einer Verkleinerung der Winkel ANB (0,70°), ANPg (0,60°) und des Wits-Wertes (1,08 mm). Der SNA-Winkel veränderte sich nicht. Die vertikale Kieferrelation (die Winkel ML/NSL, NL/NSL, ML/NL) wurde durch die Behandlung nicht beeinflusst. Die Verkleinerung des ML/NSL-Winkels ist auf normale Wachstumsveränderungen zurückzuführen. Die untere Gesichtshöhe vergrößerte sich während der Herbst-Phase signifikant (p<0,001), anterior mit einem Indexwert von 1,14 und posterior mit einem Indexwert von 1,98. Während der MB-Phase verkleinerten sich diese Indexwerte wieder leicht. Während des totalen Behandlungszeitraumes vergrößerte sich die untere Gesichtshöhe signifikant sowohl anterior mit einem Indexwert von 0,42 (p<0,05) als auch posterior mit einem Indexwert von 1,03 (p<0,01). Die Winkel zur Beschreibung der Profilkonvexität vergrößerten sich während der Herbst-Phase signifikant (p<0,001), NAPg um 2,14°, NsSnPgS um 3,92° und NsNoPgS um 2,20°. Das Hart- und Weichteilprofil wurde dadurch gerader. In der MB-Phase verkleinerten sich die Winkel wieder leicht. Während des totalen Be-handlungszeitraumes vergrößerten sich NAPg um 1,09° (p<0,01), NsSnPgS um 3,14° (p<0,001) und NsNoPgS um 1,04°(p<0,05) signifikant. Der Abstand der Oberlippe zur Esthetic-Linie wurde während der Herbst-Phase signifikant (p<0,001) um 1,63 mm größerder Abstand der Unterlippe zur Esthetic-Linie wurde signifikant (p<0,01) um 0,84 mm kleiner. In der MB-Phase vergrößerte sich der Abstand der Unterlippe wieder, der Abstand der Oberlippe veränderte sich nicht. Der Abstand der Oberlippe zur Esthetic-Linie veränderte sich während des totalen Behandlungszeitraumes signifikant (p<0,001) um 1,26 mm nach posterior, die Position der Unterlippe veränderte sich kaum. Schlussfolgernd kann festgestellt werden, dass eine Distalbissbehandlung von Erwachsenen mit der Herbst- /Multibracket-Apparatur außerordentlich erfolgreich ist und eine Alternative zu einer kieferchirurgischen Unter-kiefervorverlagerung sein könnte.The aim of this study was to assess cephalometrically the amount of dento-skeletal and facial changes during Herbst- /multibracket appliance treatment of adult Class II-11 subjects. The material consisted of 23 adult patients (4 males and 19 females) with a Class II-1. At the beginning of treatment the patients were, on average, 21,9 years old. All patients were treated with a Herbst appliance which was followed by a multibracket appliance. Lateral head films were analysed at three different times: T1: before Herbst treatment T2: after Herbst treatment T3: after multibracket treatment The changes in sagittal occlusion, sagittal and vertical jaw relationship as well as in facial height, profile convexity and lip position were assessed during three examination periods: T2-T1: Herbst phase of treatment T3-T2: multibracket phase of treatment T3-T1: total treatment period The following results were obtained: During the Herbst phase overjet correction was, on average, 9.98 mm. This was accomplished by 79 % dental and 21 % skeletal changes. Class II molar correction (6.82 mm on average) was due to 69 % dental and 31 % skeletal changes. During the following MB phase there was an insignificant relapse of the overjet and molar correction. At the end of the total treatment period, overjet and Class II molar relation were normalised in all patients. Overjet correction was, on average, 6.75 mm, due to 87 % dental and 13 % skeletal changes. Class II molar correction was, on average, 4.11 mm, resulting from 78 % dental and 22 % skeletal changes. Sagittal jaw relation was improved during the Herbst phase. An increase (p<0.001) was found in the angles SNB (1.22°) and SNPg (0.93°), thus resulting in a decrease (p<0.001) in the angles ANB (1.22°), ANPg (0.95°) and in the Wits-Appraisal (2.30 mm). During the following multibracket phase the variables relapsed insignificantly. At the end of the total treatment period an increase (p<0.001) was abserved for the angles SNB (0.82°) and SNPg (0.70°) and a decrease (p<0.001) for the angles ANB (0.70°), ANPg (0.60°) and Wits (1.08 mm). The angle SNA remained unchanged. Vertical jaw relation (ML/NSL, NL/NSL, ML/NL) did not change during the total treatment period. The reduction of ML/NSL was a result of normal growth. Lower facial height increased (p<0.001) during the Herbst phase both anteriorly (Index 1.14) and posteriorly (Index 1.98). During the MB phase the facial height relapsed insignificantly. At the end of the total treatment period lower facial height had increased significantly both anteriorly (Index 0.42, p<0.05)) and posteriorly (Index 1.03, p<0.01). The profile convexity angles increased (p<0.001) during the Herbst phase: NAPg (2.14°), NsSnPgS (3.92°) and NsNoPgS (2.20°). Thus, the profile straightened. During the MB phase, the angles relapsed insignificantly. At the end of the total treatment period the profile convexity angles had increased significantly: NAPg (1.09°, p<0.01), NsSnPgS (3.14°, p<0.001) and NsNoPgS (1.04°, p<0.05). The distance of the upper lip to the E-line increased (1.63 mm, p<0.001) during the Herbst phase, while the distance of the lower lip to the E-line decreased (0.84 mm, p<0.01). During the MB phase the position of lower lip relapsed insignificantly, the upper lip remained unchanged. At the end of the total treatment period the upper lip moved posteriorly (1.26 mm, p<0.001), while the lower lip remained unchanged. It can be concluded that treatment of adult Class II subjects with the Herbst- /multibracket appliance is successful. The treatment approach could be considered as a possible alternative to surgical mandibular advancement in borderline adult Class II subjects

A New Model for Pore Formation by Cholesterol- Dependent Cytolysins

Cholesterol Dependent Cytolysins (CDCs) are important bacterial virulence factors that form large (200–300 Å) membrane embedded pores in target cells. Currently, insights from X-ray crystallography, biophysical and single particle cryo-Electron Microscopy (cryo-EM) experiments suggest that soluble monomers first interact with the membrane surface via a C-terminal Immunoglobulin-like domain (Ig; Domain 4). Membrane bound oligomers then assemble into a prepore oligomeric form, following which the prepore assembly collapses towards the membrane surface, with concomitant release and insertion of the membrane spanning subunits. During this rearrangement it is proposed that Domain 2, a region comprising three b-strands that links the pore forming region (Domains 1 and 3) and the Ig domain, must undergo a significant yet currently undetermined, conformational change. Here we address this problem through a systematic molecular modeling and structural bioinformatics approach. Our work shows that simple rigid body rotations may account for the observed collapse of the prepore towards the membrane surface. Support for this idea comes from analysis of published cryo-EM maps of the pneumolysin pore, available crystal structures and molecular dynamics simulations. The latter data in particular reveal that Domains 1, 2 and 4 are able to undergo significant rotational movements with respect to each other. Together, our data provide new and testable insights into the mechanism of pore formation by CDCs

Domain 2-Domain 3 interface features and Molecular simulations performed.

a<p>Pairs of residues with corresponding numbering are given in brackets.</p>b<p>The number of simulations where transitions of the Domain 2/TMH2 interface occurs is given in brackets. Except for PFO I MD simulations, only one simulation is presented in this work.</p

CDC domain organisation and mechanism of pore formation.

<p>A. Crystal structure of the archetypal CDC PFO and its schematic representation. Domain 1 is coloured blue, Domain 2 coloured green, Domain 3 coloured red, orange and pink and Domain 4 coloured yellow. Together Domains 1 and 3 form the ‘head’ domain distantly related to the MACPF domain. Specific transmembrane regions include the TransMembrane Helices (TMH) 1 coloured orange and TMH2 coloured pink; the strand β5 and the undecapeptide loop are indicated. B. Current model of CDC pore formation. After the membrane binding event, monomers oligomerize into a ring-like structure (30 to 50 monomers; prepore). Upon formation of the oligomeric pore, both helical clusters insert into the transmembrane bilayer (grey bars) as two β-hairpins (orange and pink) part of a giant β-sheet barrel. Concomitantly Domain 1 is subject to a vertical collapse associated with a proposed “buckling” of Domain 2 (reviewed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003791#pcbi.1003791-Hotze1" target="_blank">[1]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003791#pcbi.1003791-Dunstone1" target="_blank">[58]</a>).</p

Pore conformation of the CDC molecule.

<p>A. Cut view of the CDC monomer in the pore conformation within the cryo-EM map (transparent surface). B. Subunits arrangement in the pore (cut view). The tetramer shown is the symmetrically modeled tetramer (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003791#s3" target="_blank">Methods</a>). Subunits have alternate colouring with Domain 2 coloured green. C. Domains 2 arrangement in the pore viewed from outside of the ring. A Domain 2 is highlighted in green sandwiched by two adjacent monomers. D. Prepore and pore conformations aligned on Domain 4. Prepore Domain 2 is in orange; pore Domain 2 is in green.</p

Twist of Domain 2 and its influence on Domain 4 orientation.

<p>A. Variations in Domain 4 orientation across the CDC family. Superposition is the same as <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003791#pcbi-1003791-g002" target="_blank">Figure 2A</a>. Only four representative structures are shown, which cover the entire range of Domain 4 orientation in CDCs. B. Coupling between Domain 2 twist and Domain 4 orientation. Domain 3 is omitted for clarity.</p