- Crystal structure of the maturation protein from bacteriophage Qβ. [Journal Article]
- JMJ Mol Biol 2017 Jan 19
- Virions of the single-stranded RNA bacteriophages contain a single copy of the maturation protein which is bound to the phage genome and is required for infectivity of the particles. The maturation p...
Virions of the single-stranded RNA bacteriophages contain a single copy of the maturation protein which is bound to the phage genome and is required for infectivity of the particles. The maturation protein mediates the adsorption of the virion to bacterial pili and subsequent release and penetration of the genome into the host cell. Here, we report a crystal structure of the maturation protein from bacteriophage Qβ. The protein has a bent, highly asymmetric shape and spans 110Å in length. Apart from small local substructures, the overall fold of the maturation protein does not resemble that of other known proteins. The protein is organized in two distinct regions, an α-helical part with a four-helix core, and a β-stranded part that contains a seven-stranded sheet in the central part and a five-stranded sheet at the tip of the protein. The Qβ maturation protein has two distinctive positively charged areas at opposite sides of the α-helical part which are involved in genomic RNA binding. The maturation protein binds to each of the surrounding coat protein dimers in the capsid differently, and the interaction is considerably weaker compared to coat protein inter-dimer contacts. The coat protein- or RNA- binding residues are not preserved among different ssRNA phage maturation proteins; instead, the distal end of the α-helical part is the evolutionary most conserved, suggesting the importance of this region for maintaining the functionality of the protein.
- Isoform-specific phosphorylation in human Hsp90β affects interaction with clients and the cochaperone Cdc37. [Journal Article]
- JMJ Mol Biol 2017 Jan 18
- Hsp90s assist maturation of many key regulators of signal transduction pathways and cellular control circuits like protein kinases and transcription factors and chaperone their stability and activity...
Hsp90s assist maturation of many key regulators of signal transduction pathways and cellular control circuits like protein kinases and transcription factors and chaperone their stability and activity. In this function Hsp90s cooperate with some 30 cochaperones and they are themselves subject to regulation by numerous posttranslational modifications. In vertebrates two major isoforms exist in the cytosol, Hsp90α and Hsp90β, which share a high degree of sequence identity and are expressed in tissue- and environmental conditions-dependent manner. We identified an isoform-specific phosphorylation site in human Hsp90β. This phosphorylation site seems to be linked to vertebrate evolution since it is not found in invertebrata but in all tetrapoda and many but not all fish species. We provide data suggesting that this phosphorylation is important for activation of Hsp90 clients like glucocorticoid receptor and a protein kinase. Replacement of the phosphorylation site by glutamate affects the conformational dynamics of Hsp90 and interaction with the kinase specific cochaperone Cdc37.
- Mechanism of action of ABC importers: conservation, divergence, and physiological adaptations. [Review]
- JMJ Mol Biol 2017 Jan 16
- The past decade has seen a remarkable surge in structural characterization of ATP Binding Cassette (ABC) transporters, which have spurred more focused functional analysis of these elaborate molecular...
The past decade has seen a remarkable surge in structural characterization of ATP Binding Cassette (ABC) transporters, which have spurred more focused functional analysis of these elaborate molecular machines. As a result, it has become increasingly apparent that there is a substantial degree of mechanistic variation between ABC transporters that function as importers, which correlates with their physiological roles. Here, we summarize recent advances in ABC importers structure-function studies and provide an explanation as to the origin of the different mechanisms of action.
- Insights into the distinct mechanisms of action of taxane and non-taxane microtubule stabilizers from cryo-EM structures. [Journal Article]
- JMJ Mol Biol 2017 Jan 16
- A number of microtubule-stabilizing agents have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We have obtai...
A number of microtubule-stabilizing agents have demonstrated or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We have obtained high-resolution (3.9-4.2Å) cryo-EM reconstructions of microtubules stabilized by the taxane-site binders Taxol and zampanolide, and by peloruside, which targets a distinct, non-taxoid pocket on β-tubulin. We find that each molecule has unique distinct structural effects on the microtubule lattice structure. Peloruside acts primarily at lateral contacts and has an effect on the "seam" of heterologous interactions, enforcing a conformation more similar to that of homologous (i.e. non-seam) contacts by which it regularizes the microtubule lattice. In contrast, binding of either Taxol or zampanolide induces microtubule heterogeneity. In doubly-bound microtubules, peloruside overrides the heterogeneity induced by Taxol-binding. Our structural analysis illustrates distinct mechanisms of these drugs for stabilizing the microtubule lattice, and is of relevance to the possible use of combinations of microtubule-stabilizing agents to regulate microtubules activity and improve therapeutic potential.
- Conformational transitions and alternating-access mechanism in the sarcoplasmic reticulum calcium pump. [Journal Article]
- JMJ Mol Biol 2017 Jan 13
- Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), a member of ...
Ion pumps are integral membrane proteins responsible for transporting ions against concentration gradients across biological membranes. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), a member of the P-type ATPases family, transports two calcium ions per hydrolyzed ATP molecule via an "alternating-access" mechanism. High-resolution crystallographic structures provide invaluable insight on the structural mechanism of the ion pumping process. However, to understand the molecular details of how ATP hydrolysis is coupled to calcium transport, it is necessary to gain knowledge about the conformational transition pathways connecting the crystallographically resolved conformations. Large-scale transitions in SERCA occur at time-scales beyond the current reach of unbiased molecular dynamics (MD) simulations. Here, we overcome this challenge by employing the string method, which represents a transition pathway as a chain-of-states linking two conformational end-points. Using a highly scalable multiscale methodology, we have determined all-atom transition pathways for three main conformational transitions responsible for the alternating-access. The present pathways provide a clear chronology of the key events underlying the active transport of calcium ions by SERCA. Important conclusions are that the conformational transition that leads to occlusion with bound ATP and calcium is highly concerted and cooperative, the phosphorylation of Asp351 causes a reorganization of the cytoplasmic domains that subsequently drives the opening of the luminal gate, and re-closing of luminal gate induces a shift in the cytoplasmic domains that subsequently enables dephosphorylation of Asp351-P. Transient residue-residue contacts during the conformational transitions predicted by the computations provide an experimental route to test the general validity of the computational pathways.
- SAXS structural studies of Dps from Deinococcus radiodurans highlights the conformation of the mobile N-terminal extensions. [Journal Article]
- JMJ Mol Biol 2017 Jan 11
- The radiation resistant bacterium Deinococcus radiodurans contains two DNA binding proteins from starved cells (Dps): Dps1 (DR2263) and Dps2 (DRB0092). These are suggested to play a role in DNA inter...
The radiation resistant bacterium Deinococcus radiodurans contains two DNA binding proteins from starved cells (Dps): Dps1 (DR2263) and Dps2 (DRB0092). These are suggested to play a role in DNA interaction, manganese and iron storage. The proteins assemble as a conserved dodecameric structure with structurally uncharacterized N-terminal extensions. In the case of DrDps1 these extensions have been proposed to be involved in DNA interactions, while in DrDps2 their function has yet to be established. The reported data reveals the relative position of the N-terminal extensions to the dodecameric sphere in solution for both Dps. The low resolution small angle X-ray scattering (SAXS) results show that the N-terminal extensions protrude from the spherical shell of both proteins. The SAXS envelope of a truncated form of DrDps1 without the N-terminal extensions appears as a dodecameric sphere, contrasting strongly with the protrusions observed in the full length models. The effect of iron incorporation into DrDps2 was investigated by static and stopped-flow SAXS measurements, revealing dynamic structural changes upon iron binding and core formation, as reflected by a quick alteration of its radius of gyration. The truncated and full length versions of DrDps were also compared on the basis of their interaction with DNA to analyse functional roles of the N-terminal extensions. DrDps1 N-terminal protrusions appear to be involved directly in interacting with DNA, whilst those from DrDps2 are indirectly associated with DNA binding. Furthermore, detection of DrDps2 in the D. radiodurans membrane fraction suggests that the N-terminal of the protein interacts with the membrane.
- Sensing Membrane Curvature in Macroautophagy. [Review]
- JMJ Mol Biol 2017 Jan 11
- In response to intracellular stress events ranging from starvation to pathogen invasion, the cell activates one or more forms of macroautophagy. The key event in these related pathways is the de novo...
In response to intracellular stress events ranging from starvation to pathogen invasion, the cell activates one or more forms of macroautophagy. The key event in these related pathways is the de novo formation of a new organelle called the autophagosome, which either surrounds and sequesters random portions of the cytoplasm or selectively targets individual intracellular challenges. Thus, the autophagosome is a flexible membrane platform with dimensions that ultimately depend upon the target cargo. The intermediate membrane, termed the phagophore or isolation membrane, is a cup-like structure with a clear concave face and a highly curved rim. The phagophore is largely devoid of integral membrane proteins; thus, its shape and size are governed by peripherally associated membrane proteins and possibly by the lipid composition of the membrane itself. Growth along the phagophore rim marks the progress of both organelle expansion and ultimately organelle closure around a particular cargo. These two properties, a reliance on peripheral membrane proteins and a structurally distinct membrane architecture, suggest that the ability to target or manipulate membrane curvature might be an essential activity of proteins functioning in this pathway. In this review, we discuss the extent to which membranes are naturally curved at each of the cellular sites believed to engage in autophagosome formation, review basic mechanisms used to sense this curvature, and then summarize the existing literature concerning which autophagy proteins are capable of curvature recognition.
- Three-dimensional structure of full-length NtrX, an unusual member of the NtrC family of response regulators. [Journal Article]
- JMJ Mol Biol 2017 Jan 11
- Bacteria sense and adapt to environmental changes using two-component systems (TCS). These signaling pathways are formed by a histidine kinase (HK) that phosphorylates a response regulator (RR), whic...
Bacteria sense and adapt to environmental changes using two-component systems (TCS). These signaling pathways are formed by a histidine kinase (HK) that phosphorylates a response regulator (RR), which finally modulates the transcription of target genes. The bacterium Brucella abortus codes for a TCS formed by the HK NtrY and the RR NtrX that participates in sensing low oxygen tension and in generating an adaptive response. NtrX is a modular protein with REC, AAA+ and DNA binding domains, an architecture that classifies it among the NtrC subfamily of RRs. However, it lacks the signature GAFTGA motif that is essential for activating transcription by the mechanism proposed for canonical members of this subfamily. In this article we present the first crystal structure of full-length NtrX, which is also the first structure of a full-length NtrC-like RR with all the domains solved, showing that the protein is structurally similar to other members of the subfamily. We also report that NtrX binds nucleotides and the structures of the protein bound to ATP and ADP. Despite binding ATP, NtrX does not have ATPase activity and does not form oligomers in response to phosphorylation or nucleotide binding. We also identify a nucleotide sequence recognized by NtrX that allows it to bind to a promoter region that regulates its own transcription and to establish a negative feedback mechanism to modulate its expression. Overall, this article provides a detailed description of the NtrX RR and supports that it functions by a mechanism different to classical NtrC-like RRs.
- Molecular Mechanisms of Autophagy-Part B. [Editorial]
- JMJ Mol Biol 2017 Jan 11
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- Bacteriophage SPP1 pac Cleavage: A Precise Cut without Sequence Specificity Requirement. [Journal Article]
- JMJ Mol Biol 2017 Jan 09
- In many tailed bacteriophages, DNA packaging is initiated by recognition and cleavage of a specific sequence pac by the small (TerS) and large (TerL) terminase subunits. It was previously shown that ...
In many tailed bacteriophages, DNA packaging is initiated by recognition and cleavage of a specific sequence pac by the small (TerS) and large (TerL) terminase subunits. It was previously shown that the SPP1 pac region has two sequences where TerS binds (pacR and pacL), flanking the segment where TerL cleaves the SPP1 DNA (pacC). However, the pac-specific sequences required to achieve this endonucleolytic cut were not established. Their characterization is essential to understand the underlying mechanism. We show that the pacR sequence localized within 35bp downstream of the pac cut can be extensively degenerated, including its c1 and c2 repeats, and that only a disruption of a 5-bp polyadenine tract impairs the pac cleavage. This result together with deletion analysis of pacL shows that the specific DNA sequences required for targeting the terminase for pac cleavage are considerably shorter than the large region bound by TerS. Furthermore, extensive degeneration of the 6-bp target sequence within pacC where pac cleavage occurs reveals that TerL maintains, remarkably, its precise position of cleavage. Studies with SPP1-related phages show the conservation of the cut position, irrespective of the sequence variation in pacC and in pacR or the changes in pacL-pacC distance. Mechanistically, our data are compatible with a model in which TerS interactions with part of the pacL sequence and a poly-A tract in pacR are sufficient to orient very accurately the TerL nuclease to a defined pacC position. They also demonstrate that the resulting precise cut at pacC is independent of the targeted DNA sequence.