Monomerization alters the dynamics of the lid region in Campylobacter jejuni CstII: an MD simulation study

CstII, a bifunctional (α2,3/8) sialyltransferase from Campylobacter jejuni, is a homotetramer. It has been reported that mutation of the interface residues Phe121 (F121D) or Tyr125 (Y125Q) leads to monomerization and partial loss of enzyme activity, without any change in the secondary or tertiary structures. MD simulations of both tetramer and monomer, with and without bound donor substrate, were performed for the two mutants and WT to understand the reasons for partial loss of activity due to monomerization since the active site is located within each monomer. RMSF values were found to correlate with the crystallographic B-factor values indicating that the simulations are able to capture the flexibility of the molecule effectively. There were no gross changes in either the secondary or tertiary structure of the proteins during MD simulations. However, interface is destabilized by the mutations, and more importantly the flexibility of the lid region (Gly152-Lys190) is affected. The lid region accesses three major conformations named as open, intermediate, and closed conformations. In both Y121Q and F121D mutants, the closed conformation is accessed predominantly. In this conformation, the catalytic base His188 is also displaced. Normal mode analysis also revealed differences in the lid movement in tetramer and monomer. This provides a possible explanation for the partial loss of enzyme activity in both interface mutants. The lid region controls the traffic of substrates and products in and out of the active site, and the dynamics of this region is regulated by tetramerization. Thus, this study provides valuable insights into the role of loop dynamics in enzyme activity of CstII.     

Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

Wie V?gel ihr Auge schützen: Zur Arbeitsteilung von Oberlid, Unterlid und Nickhaut

Zusammenfassung Vögel schließen ihre Augen im Schlaf in einer für die großen Taxa typischen Weise. Entweder geht das Unterlid hoch wie bei der Mehrzahl der Arten, oder das Oberlid bewegt sich abwärts (Psittaciformes, Trochili), oder aber beide Lider schließen die Lidspalte (Strigiformes, Caprimulgi). Solche Kenntnis fehlt von den meisten Ordnungen, oder die Handbücher geben falsche oder widersprüchliche Information. Neben dem tonischen, schlafbegleitenden Augenschluss bewegen Vögel im Wachzustand eines oder beide Lider phasisch und meist schnell. Dieser häufige Lidschlag ist durch ein anderes Bewegungsmuster und durch eine andere Funktion gekennzeichnet. Photodokumente und genaue Beobachtungen führen erstmals zu einer funktionellen Deutung, der zufolge der Lidschlag das Auge mechanisch schützt. Droht dem Auge von vorn oder von oben eine potentielle Schädigung, so schließt das Oberlid bei Tauben, Eulen und Singvögeln, im Sprühwasser gleichzeitig auch das Unterlid (Cinclus). Der unabweisbarste Beleg stammt aus dem Vergleich des Aufpickens dorniger, sperriger Beuteinsekten mit Oberlidschluss gegenüber dem Aufnehmen harmloser Beeren ohne jede Lidbewegung (Gallicolumba). Weiter ist die Antwort des Oberlids, anders als beim Unterlid, öfter seitengerecht reizorientiert, so dass die Bewegung einseitig sein kann. Zudem kann der Schluss des Oberlids auch bei stationärem (Feind-)reiz seitenweise alternieren (Otus). Ausnahmsweise tritt eine adaptive Asymmetrie auch während kurzer Zeiten der Augenöffnung zum Spähen nach Feinden im Schlaf auf, und zwar hier beim Unterlid der bedrohten Seite (Anas).Eine neue Funktion wird auch dem Schlag der Nickhaut (Membrana nictitans) zugeschrieben. Traditionell als die Cornea reinhaltendes Organ gesehen, dient auch sie dem mechanischen Schutz des Auges. Auch sie kann seitenrichtig reizorientiert schlagen, doch ist hierüber wenig bekannt. Dieselben Reize, die den Lidschlag auslösen, können bei anderen Arten die Nickhaut schlagen lassen. Ihre Schlagrate ist schwierig zu messen, da viele Schläge (nur?) mit denen des Oberlides zusammenfallen und so verborgen bleiben (Otus). Diese Synchronie ist mit keiner der bisher vorgeschlagenen Funktionen erklärbar, ebenso wenig wie die verborgenen Schläge bei tonischem Augenschluss (Passer).Die Annahme einer Ausschaltung störender Sinnesinformation, z.B. während rascher Kopfbewegungen, durch die Nickhaut lässt sich aus vier Gründen verwerfen. Die Zunahme der Schlagrate während des Feindalarms (Ficedula) bleibt funktionell unerklärt.In einer bei Vögeln einzigartigen Weise schützt der Samtkleiber (Sitta azurea) sein Auge durch Zusammenziehen des nackten Augenrings (Lidblende), wenn er rücklings an der Unterseite von Ästen nahrungssuchend einem ständigen Regen von losgelösten Rindenteilchen u. ä. ausgesetzt ist.Sekundär haben sich die Bewegungen eines oder beider Lider oder aber der Nickhaut zu optischen Signalen entwickelt, und zwar durch kontrastierende Feder- oder Nickhautfärbung. Die betreffenden blitzschnell aufleuchtenden Signale sind an den Paarpartner (Cinclus, Corvidae,Cepphus), an mögliche Feinde (Anas) oder an bisher unbekannte Empfänger gesichtet (Ficedula).

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On how birds protect their eyes: division of labour between the upper lid, lower lid and the nictitating membrane

Summary Birds close their eyes during sleep in various taxon-specific ways. Either the lower lid moves up as in the majority of species including the Anseres, Accipitres, Falconiformes, Galli, Charadrioidea, Columbiformes, and Oscines; or the upper lid moves down (Psittaciformes, Trochili), or both lids close the eye as in Strigiformes and Caprimulgi. Such information is absent for most orders, or the handbooks provide wrong or conflicting information. Beside the tonic, sleep-related eye closure, birds move one or both lids in a phasic, usually swift mode when awake. These frequent lid movements are typified by their different co-ordination and function. Photographic and observational evidence strongly suggests mechanical protection of the eye as a novel function (where this had not been proposed previously). When an impact from any object is imminent from in front of or above the head, the upper lid shuts in pigeons, owls and oscines, and with water splashing, the lower lid as well (Cinclus). The most convincing evidence for mechanical protection comes from the deployment of the upper lid during the picking up of spiny insect prey as compared to easy-to-swallow berries, when both lids stay at rest (Gallicolumba).Further, the response of the upper lid is more stimulus-oriented so that both upper lids move asymmetrically. But there is also a unilateral, alternating winking of the upper lids when causative (predator) stimuli remain stationary. This never occurs with the lower lids (Otus). As an exception, an adaptive asymmetry occurs during brief phases of unilateral scanning interrupting sleep, designed to detect approaching predators. This scanning involves the lower lid (Anas).A new function is also attributed to the beating of the nictitating membrane (Membrana nictitans). Traditionally viewed as a cleaning device it also serves to protect the eye from mechanical impact, and it also can be tuned to the side from where danger is threatening, though by and large there is a dearth of information from avian taxa. The non-visually elicited action of the membrane seems always to be bilateral (Falco, Harpia). The very stimuli eliciting the blinking of a lid can, in different species, trigger the beat of the membrane, and can cause it to move tonically (Falco). The membrane beats at a rate difficult to measure since many of its beats coincide with the blinking of the upper lid and thus remain hidden (Otus). This coincidence is difficult to account for by any function discussed so far, as are the many hidden beats during tonic eye closure with the lids (Passer).The hypothesis according to which the action of the membrane is filtering out undesirable retinal stimulation during e.g. rapid head movements is dismissed on four different grounds. The increase of the membrane activity during predator alarm (Ficedula) is functionally unaccounted for.In a fashion unique among birds, the Blue Nuthatch (Sitta azurea) protects its eyes by contracting the naked skin surrounding the eye, thereby minimizing the exposure of the cornea; during foraging along the underside of branches, a continual rain of bark particles and debris jeopardizes unimpeded vision.Secondarily, one or both lids or the nictitating membrane have taken on the function of optic signals by virtue of contrasting feather colour or coloration. The phasic (flashing) signal movements involved are directed at the pair mate (Cinclus, Corvidae,Cepphus), predators (Anas) or at unknown parties (Ficedula).

Dies ist Veröffentlichung Nr. 29 des Philippine Endemic Species Conservation Project der Zoologischen Gesellschaft Frankfurt.     

Solvent-induced lid opening in lipases: A molecular dynamics study

In most lipases, a mobile lid covers the substrate binding site. In this closed structure, the lipase is assumed to be inactive. Upon activation of the lipase by contact with a hydrophobic solvent or at a hydrophobic interface, the lid opens. In its open structure, the substrate binding site is accessible and the lipase is active. The molecular mechanism of this interfacial activation was studied for three lipases (from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosa) by multiple molecular dynamics simulations for 25 ns without applying restraints or external forces. As initial structures of the simulations, the closed and open structures of the lipases were used. Both the closed and the open structure were simulated in water and in an organic solvent, toluene. In simulations of the closed lipases in water, no conformational transition was observed. However, in three independent simulations of the closed lipases in toluene the lid gradually opened. Thus, pathways of the conformational transitions were investigated and possible kinetic bottlenecks were suggested. The open structures in toluene were stable, but in water the lid of all three lipases moved towards the closed structure and partially unfolded. Thus, in all three lipases opening and closing was driven by the solvent and independent of a bound substrate molecule.     

Base-CP proteasome can serve as a platform for stepwise lid formation

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.     

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics

The p53‐MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein‐ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit‐solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb‐ildn, ff99sb‐ildn‐nmr, ff99sb‐ildn‐phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200‐ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo‐like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands. Proteins 2015; 83:1665–1676. © 2015 Wiley Periodicals, Inc.     

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

On the functional role of Arg172 in substrate binding and allosteric transition in Escherichia coli glucosamine-6-phosphate deaminase

Glucosamine-6-phosphate deaminase from Escherichia coli (EC 3.5.99.6) is an allosteric enzyme, activated by N-acetylglucosamine 6-phosphate, which converts glucosamine-6-phosphate into fructose 6-phosphate and ammonia. X-ray crystallographic structural models have showed that Arg172 and Lys208, together with the segment 41-44 of the main chain backbone, are involved in binding the substrate phospho group when the enzyme is in the R activated state. A set of mutants of the enzyme involving the targeted residues were constructed to analyze the role of Arg172 and Lys208 in deaminase allosteric function. The mutant enzymes were characterized by kinetic, chemical, and spectrometric methods, revealing conspicuous changes in their allosteric properties. The study of these mutants indicated that Arg172 which is located in the highly flexible motif 158-187 forming the active site lid has a specific role in binding the substrate to the enzyme in the T state. The possible role of this interaction in the conformational coupling of the active and the allosteric sites is discussed.     

Zipping and Unzipping of Adenylate Kinase: Atomistic Insights into the Ensemble of Open ↔ Closed Transitions

Adenylate kinase (AdK), a phosphotransferase enzyme, plays an important role in cellular energy homeostasis. It undergoes a large conformational change between an open and a closed state, even in the absence of substrate. We investigate the apo-AdK transition at the atomic level both with free-energy calculations and with our new dynamic importance sampling (DIMS) molecular dynamics method. DIMS is shown to sample biologically relevant conformations as verified by comparing an ensemble of hundreds of DIMS transitions to AdK crystal structure intermediates. The simulations reveal in atomic detail how hinge regions partially and intermittently unfold during the transition. Conserved salt bridges are seen to have important structural and dynamic roles; in particular, four ionic bonds that open in a sequential, zipper-like fashion and, thus, dominate the free-energy landscape of the transition are identified. Transitions between the closed and open conformations only have to overcome moderate free-energy barriers. Unexpectedly, the closed state and the open state encompass broad free-energy basins that contain conformations differing in domain hinge motions by up to 40°. The significance of these extended states is discussed in relation to recent experimental Förster resonance energy transfer measurements. Taken together, these results demonstrate how a small number of cooperative key interactions can shape the overall dynamics of an enzyme and suggest an “all-or-nothing” mechanism for the opening and closing of AdK. Our efficient DIMS molecular dynamics computer simulation approach can provide a detailed picture of a functionally important macromolecular transition and thus help to interpret and suggest experiments to probe the conformational landscape of dynamic proteins such as AdK.     

脂肪酶活性部位上的盖与界面活性

脂肪酶是一种非常重要的水解酶,在工业催化、医药和科学研究等领域中有广泛应用. 大部分脂肪酶的活性部位上方有一段被称为“盖”的α-螺旋,这种盖赋予脂肪酶在水/油界面上有特殊的催化活性,界面活性.而在单一水相或油相中却表现出低活性或无活性.界面活性与盖的组成、大小、构象及其存在环境等密切相关,探明盖与脂肪酶界面活性的关系对于脂肪酶的开发和利用是非常关键的.因此,长期以来人们对盖在脂肪酶催化作用中所扮演的角色进行了孜孜不倦的探索.本文从盖的构象、移动、组成和删除等方面综述了其对脂肪酶催化作用的影响,期望对人们认识脂肪酶盖与其催化作用之间的关系有一定的帮助.