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Journal of molecular biology [journal]
- The Crystal Structure of the Human Titin:Obscurin Complex Reveals a Conserved Yet Specific Muscle M-band Zipper Module. [JOURNAL ARTICLE]
- J Mol Biol 2014 Dec 6.
M10 is the most C-terminal immunoglobulin (Ig) domain of the giant protein titin and a frequent target of disease-linked mutations. Currently, it is the only known muscle Ig-domain able to interact with two alternative ligands - obscurin and obscurin-like-1 (Obsl1) - in different sarcomeric subregions. Obscurin and Obsl1 use their homologous N-terminal Ig domain (O1 in obscurin and OL1 in Obsl1) to bind M10 in a mutually exclusive manner. We present here the X-ray structure of the human titin:obscurin M10:O1 complex extending our previous work on the M10:OL1 interaction. Similar to M10:OL1, the M10:O1 complex displays a chevron-shaped antiparallel Ig-Ig architecture held together by a conserved molecular interface, which we validated by isothermal titration calorimetry and sorting experiments in neonatal rat cardiomyocytes (NRCs). O1 although structurally related to OL1 and M10, both members of the I-set Ig family, presents an intriguing switch of its βA' strand. This leads to structural differences between the complexes, particularly, for the 'open-side' of the chevron-shaped assembly. A bioinformatics analysis reveals that the βA'-switch observed for O1 is rare and that it is involved in mediating protein-protein interactions. Molecular Dynamics simulations also suggest that this topological alteration substantially increases local flexibility compared to the conventional I-set Ig domains. The O1/OL1 Ig domains are candidate discriminatory structural modules potentially directing the binding of specific additional partners at the M-band. Cellular sorting experiments in NRCs are consistent with the view that the titin:obscurin/Obsl1 complexes might be a platform for higher order interactions.
- Structure of an APC3-APC16 complex: Insights into assembly of the Anaphase Promoting Complex/Cyclosome. [JOURNAL ARTICLE]
- J Mol Biol 2014 Dec 6.
The Anaphase Promoting Complex/Cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the "Platform" centers around a cullin-RING-like E3 ligase catalytic core; the "Arc Lamp" is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-twofold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7, and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR-tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer, and together with biochemistry and prior data explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR-tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.
- Mapping the gating and permeation pathways in the voltage-gated proton channel Hv1. [JOURNAL ARTICLE]
- J Mol Biol 2014 Dec 3.
Voltage-gated proton channels (Hv1) are ubiquitous throughout nature and are implicated in numerous physiological processes. The gene encoding for Hv1 however was only identified in 2006. The lack of sufficient structural information of this channel has hampered the understanding of the molecular mechanism of channel activation and proton permeation. This study uses both simulation and experimental approaches to further develop existing models of the Hv1 channel. Our study provides insights into features of channel gating and proton permeation pathway. We compare open- and closed-state structures developed previously with a recent crystal structure that traps the channel in a presumably closed state. Insights into gating pathways were provided using a combination of all-atom MD simulations with a swarm-of-trajectories with the string method for extensive transition path sampling and evolution. A detailed residue-residue interaction profile and a hydration profile were studied to map the gating pathway in this channel. In particular it allows us to identify potential intermediate states and compare them to the experimentally observed crystal structure of Takeshita et al . The mechanisms governing ion transport in the WT and mutant Hv1 channels were studied by a combination of electrophysiological recordings and free energy simulations. With these results we were able to further refine ideas about the location and function of the selectivity filter. The refined structural models will be essential for future investigations of this channel and the development of new drugs targeting cellular proton transport.
- An Improved Single-chain Fab Platform for Efficient Display and Recombinant Expression. [JOURNAL ARTICLE]
- J Mol Biol 2014 Dec 3.
Antibody phage display libraries combined with high-throughput selections have recently demonstrated tremendous promise to create the next generation of renewable, recombinant antibodies to study proteins and their many post-translational modification states; however many challenges still remain, such as optimized antibody scaffolds. Recently, a single-chain Fab (scFab) format, in which the carboxy-terminus of the light chain is linked to the amino-terminus of the heavy chain, was described to potentially combine the high display levels of a single-chain Fv with the high stability of purified Fabs. However, this format required removal of the interchain disulfide bond to achieve modest display levels and subsequent bacterial expression resulted in high levels of aggregated scFab, hindering further use of scFabs. Here, we developed an improved scFab format that retains the interchain disulfide bond by increasing the linker length between the light and heavy chains to improve display and bacterial expression levels to 1-3mg per liter. Furthermore, rerouting of the scFab to the co-translational signal recognition particle (SRP) pathway combined with reengineering of the signal peptide sequence results in display levels 24-fold above the original scFab format and 3-fold above parent Fab levels. This optimized scFab scaffold can be easily reformatted in a single step for expression in a bacterial or mammalian host to produce stable (81°C Tm), predominantly monomeric (>90%) antibodies at a high yield. Ultimately, this new scFab format will advance high-throughput antibody generation platforms to discover the next generation of research and therapeutic antibodies.
- Specificity Determinants in Small Multidrug Transporters. [JOURNAL ARTICLE]
- J Mol Biol 2014 Dec 3.
Multiple-antibiotic resistance has become a major global public health concern, and to overcome this problem, it is necessary to understand the resistance mechanisms that allow survival of the microorganisms at the molecular level. One mechanism responsible for such resistance involves active removal of the antibiotic from the pathogen cell by MDTs (multidrug transporters). A prominent MDT feature is their high polyspecificity allowing for a single transporter to confer resistance against a range of drugs. Here we present the molecular mechanism underlying substrate recognition in EmrE, a small MDT from Escherichia coli. EmrE is known to have a substrate preference for aromatic, cationic compounds, such as methyl viologen (MV(2+)). In this work, we use a combined bioinformatic and biochemical approach to identify one of the major molecular determinants involved in MV(2+) transport and resistance. Replacement of an Ala residue with Ser in weakly resistant SMRs from Bacillus pertussis and Mycobacterium tuberculosis enables them to provide robust resistance to MV(2+) and to transport MV(2+) and has negligible effects on the interaction with other substrates. This shows that the residue identified herein is uniquely positioned in the binding site so as to be exclusively involved in the mediating of MV(2+) transport and resistance, both in EmrE and in other homologues. This work provides clues toward uncovering how specificity is achieved within the binding pocket of a polyspecific transporter that may open new possibilities as to how these transporters can be manipulated to bind a designed set of drugs.
- Permeation and Dynamics of an Open-Activated TRPV1 Channel. [JOURNAL ARTICLE]
- J Mol Biol 2014 Dec 3.
Transient receptor potential (TRP) ion channels constitute a large and diverse protein family, found in yeast and widespread in the animal kingdom. TRP channels work as sensors for a wide range of cellular and environmental signals. Understanding how these channels respond to physical and chemical stimuli has been hindered by the limited structural information available until now. The three-dimensional structure of the vanilloid receptor 1 (TRPV1) was recently determined by single particle electron cryo-microscopy, offering for the first time the opportunity to explore ionic conduction in TRP channels at atomic detail. In this study, we present molecular dynamics simulations of the open-activated pore domain of TRPV1 in the presence of three cationic species: Na(+), Ca(2+) and K(+). The dynamics of these ions while interacting with the channel pore allowed us to rationalize their permeation mechanism in terms of a pathway involving three binding sites at the intracellular cavity, as well as the extracellular and intracellular entrance of the selectivity filter. Furthermore, conformational analysis of the pore in the presence of these ions reveals specific ion-mediated structural changes in the selectivity filter, which influences the permeability properties of the TRPV1 channel.
- Bidirectional promoters of insects: genome-wide comparison, evolutionary implication and influence on gene expression. [JOURNAL ARTICLE]
- J Mol Biol 2014 Nov 15.
Bidirectional promoters are widespread in insect genomes. By analyzing 23 insect species, we show that the frequency of bidirectional gene pairs varies according to genome compactness and density of genes among the genomes. The expected density of bidirectional genes based on number of genes per megabase of genome explains the observed density indicating that bidirectional pairing of genes may be due to random event. We identified specific transcription factor binding motifs that are enriched in bidirectional promoters across insect species. Furthermore, we observed that bidirectional promoters may act as transcriptional hotspots in insect genomes where protein coding genes tend to aggregate around bidirectional promoters in significantly biased (p <0.001) manner compared to unidirectional promoters. Natural selection seems to have an association with the extent of bidirectionality of genes among the species. The rate of non-synonymous to synonymous (dN/dS) changes shows a second-order polynomial distribution with bidirectionality between species indicating that bidirectionality is dependent upon evolutionary pressure acting on the genomes. Analysis of genome-wide microarray expression data of multiple insect species suggested that bidirectionality have a similar association with transcriptome variation across species. Furthermore, bidirectional promoters show significant association with correlated expression of the divergent gene pairs depending upon their motif composition. Analysis of gene ontology further showed that bidirectional genes tend to have a common association with 'binding' (including ion binding, nucleotide binding and protein binding) related functions across genomes. Such functional constraint of bidirectional genes may explain their widespread persistence in genome of diverse insect species.
- The Activity and Stability of the Intrinsically Disordered Cip/Kip Protein Family AreRegulated by Non-Receptor TyrosineKinases. [JOURNAL ARTICLE]
- J Mol Biol 2014 Nov 20.
The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21(Cip1), p27(Kip1) and p57(Kip2). Their kinase inhibitory activities are mediated by a homologous N-terminal kinaseinhibitory domain. The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the kinase inhibitory domain and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, as well as its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation.
- Movement of Elongation Factor G between Compact and Extended Conformations. [JOURNAL ARTICLE]
- J Mol Biol 2014 Nov 15.
Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer. Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pretranslocation ribosomes or with posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to but likely precedes both GTP hydrolysis and mRNA/tRNA translocation.
- Distinct Features of Cap Binding by eIF4E1b Proteins. [JOURNAL ARTICLE]
- J Mol Biol 2014 Nov 15.
eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m(7)GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α+β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N(7) of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N(7) position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.