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Journal of molecular biology [journal]
- Antigen Translocation Machineries in Adaptive Immunity and Viral Immune Evasion. [REVIEW]
- J Mol Biol 2014 Sep 12.
Protein homeostasis results in a steady supply of peptides, which are further degraded to fuel protein synthesis or metabolic needs of the cell. In higher vertebrates, a small fraction of the resulting peptidome, however, is translocated into the ER lumen by the transporter associated with antigen processing (TAP). Antigenic peptides are guided to major histocompatibility complex class I (MHC I) molecules and are finally displayed on the cell surface, where they mount an adaptive immune response against viral infected or malignantly transformed cells. Here, we review the structural organization and the molecular mechanism of this specialized antigen translocon. We discuss how the ATP-binding cassette (ABC) transporter TAP communicates and cooperates within the multi-component peptide-loading machinery, mediating the proper assembly and editing of kinetically stable peptide/MHC I complexes. In light of its important role within the MHC I antigen processing pathway, TAP is a prime target for viral immune evasion strategies, and we summarize how this antigen translocation machinery is sabotaged by viral factors. Finally, we compare TAP with other ABC systems that facilitate peptide translocation.
- Structural analysis of the human Fibroblast Growth Factor Receptor 4 Kinase. [JOURNAL ARTICLE]
- J Mol Biol 2014 Sep 11.
The family of Fibroblast Growth Factor Receptors (FGFRs) plays an important and well characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer e.g. breast cancer or rhabdomyosarcoma. Whereas FGFRs 1-3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I-inhibitor Dovitinib and the type II-inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4-inhibitors.
- A Cutinase from Trichoderma reesei with a Lid-covered Active Site and Kinetic Properties of True Lipases. [JOURNAL ARTICLE]
- J Mol Biol 2014 Sep 11.
Cutinases belong to the α/β hydrolase fold family of enzymes and degrade cutin as well as various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated as well as soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr cutinase is its optimal activity at acidic pH. Furthermore, Tr cutinase, in contrast with classical cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr cutinase to be a member of a new group of lipolytic enzymes resembling cutinases but with kinetic and structural features of true lipases and a heightened specificity for long chain triglycerides.
- Editorial for Special Issue: Insights into Molecular Mechanisms of Microbiota. [EDITORIAL]
- J Mol Biol 2014 Sep 10.
- Ion Channel Engineering: Perspectives and Strategies. [REVIEW]
- J Mol Biol 2014 Sep 6.
Ion channels facilitate the passive movement of ions down an electrochemical gradient and across lipid bilayers in cells. This phenomenon is essential for life, and underlies many critical homeostatic processes in cells. Ion channels are diverse and differ with respect to how they open and close (gating), and their ionic conductance/selectivity (permeation). Fundamental understanding of ion channel structure-function mechanisms, their physiological roles, how their dysfunction leads to disease, their utility as biosensors, and development of novel molecules to modulate their activity are important and active research frontiers. In this review, we focus on ion-channel engineering approaches that have been applied to investigate these aspects of ion channel function, with a major emphasis on voltage-gated ion channels.
- "Breaking up is hard to do" The formation and resolution of sister chromatid intertwines. [REVIEW]
- J Mol Biol 2014 Sep 3.
The absolute necessity to resolve every intertwine between the two strands of the DNA double helix provides a massive challenge to the cellular processes that duplicate and segregate chromosomes. Although the overwhelming majority of intertwines between the parental DNA strands are resolved during DNA replication, there are numerous chromosomal contexts where some intertwining is maintained into mitosis. These mitotic sister chromatid intertwines (SCI) can be found as short regions of unreplicated DNA, fully replicated and intertwined sister chromatids - commonly referred to as DNA catenation - and as sister chromatid linkages generated by homologous recombination associated processes. Several overlapping mechanisms, including intra-chromosomal compaction, topoisomerase action and Holliday junction resolvases ensure all SCIs are removed before they can prevent normal chromosome segregation. Here, I discuss why some DNA intertwines persist into mitosis and review our current knowledge of the SCI resolution mechanisms that are employed in both prokaryotes and eukaryotes, including how deregulating SCI formation during DNA replication or disrupting the resolution processes may contribute to aneuploidy in cancer.
- Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway. [JOURNAL ARTICLE]
- J Mol Biol 2014 Sep 1.
The small GTPase protein Sar1 is known to be involved in both the initiation of COPII coated vesicle formation and scission of the nascent vesicle from the ER. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.
- A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase. [JOURNAL ARTICLE]
- J Mol Biol 2014 Sep 2.
Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a "wedge" that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.
- Characterization of the Grp94/OS-9 chaperone-lectin complex. [JOURNAL ARTICLE]
- J Mol Biol 2014 Sep 2.
Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER associated degradation (ERAD) quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins (TMPs). To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal MRH lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains 'mammalian-specific insets' that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate, and additionally hints at a plausible biological role for the Grp94/OS-9 complex.
- Nuclear organization changes and the epigenetic silencing of FLC during vernalization. [REVIEW]
- J Mol Biol 2014 Aug 30.
Changes in nuclear organization are considered an important complement to trans-acting factors, histone modifications and non-coding RNAs in robust and stable epigenetic silencing. However, how these multiple layers interconnect mechanistically to reinforce each other's activity is still unclear. A system providing long timescales facilitating analysis of these interconnections is vernalization. This involves the Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC) that occurs as Arabidopsis plants are exposed to prolonged cold. Analysis of changes in nuclear organization during vernalization has revealed that disruption of a gene loop and physical clustering of FLC loci are part of the vernalization mechanism. These events occur at different times and thus contribute to distinct aspects of the silencing mechanism. The physical clustering of FLC loci is tightly correlated with the accumulation of specific Polycomb complexes/H3K27me3 at a localized intragenic site during the cold. Since the quantitative nature of vernalization is a reflection of a bistable cell-autonomous switch in an increasing number of cells this correlation suggests a tight connection between the switching mechanism and changes in nuclear organization. This integrated picture is likely to be informative for many epigenetic mechanisms.