High affinity binding of the receptor-associated protein D1D2 domains with the LDL receptor-related protein (LRP1) involves bivalent complex formation: Critical roles of lysine 60 and 191. [Molecular Biophysics]

July 11th, 2016 by Prasad, J. M., Young, P. A., Strickland, D. K.

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three domain protein that contains two high affinity binding sites for LRP1: one located within domains 1 and 2, and one located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2/LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor.
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NEDD4L Catalyzes Ubiquitination of PIK3CA and Regulates PI3K-AKT Signaling [Signal Transduction]

June 23rd, 2016 by Wang, Z., Dang, T., Liu, T., Chen, S., Li, L., Huang, S., Fang, M.

Oncogenic PIK3CA (p110α), the catalytic subunit of class IA PI3K, plays a major role in PI3K related cancer progression. The mechanisms underlying the dynamic regulation of PIK3CA protein levels remain unknown. Here we demonstrated that PIK3CA is regulated by polyubiquitination. We identified NEDD4L as the E3 ligase that catalyzes PIK3CA polyubiquitination, leading to its proteasome-dependent degradation. NEDD4L ubiquitinates both the free and regulatory subunit-bound PIK3CA, but does not ubiquitinate the regulatory subunit of PI3K. Overexpression of NEDD4L accelerates the turnover rate of PIK3CA whereas suppression of NEDD4L results in not only the accumulation of PIK3CA but also a paradoxical decrease of AKT activation. Thus, we propose that NEDD4L negatively regulates PIK3CA protein levels via ubiquitination and is required for the maintenance of PI3K-AKT signaling pathway.

Sialylation of GPI Anchors of Mammalian Prions is Regulated in a Host-, Tissue- and Cell-Specific Manner [Glycobiology and Extracellular Matrices]

June 17th, 2016 by Katorcha, E., Srivastava, S., Klimova, N., Baskakov, I. V.

Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrPC. PrPC is posttranslationally modified with N-linked glycans and a sialylated glycosylinositol phospholipid anchor (GPI). Conformational conversion of PrPC give rise to glycosylated and GPI-anchored PrPSc. The question of the sialylation status of GPIs within PrPSc has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrPSc with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrPC with sialylo-GPIs could be recruited into PrPSc, whereas PrPC with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed sialylation status of GPIs within PrPSc generated in brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrPC with both sialo- and asialo-GPIs is a common feature of PrPSc. The mixtures of sialo- and asialo-GPIs were observed in PrPSc universally regardless of prion strain, as well as host, tissue or type of cells that produced PrPSc. Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue and cell type, but not prion strain. In summary, the current study found no strain-specific preferences for selecting PrPC with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrPSc is regulated in a cell-, tissue- or host-specific manner, and is likely to be determined by the specifics of GPI biosynthesis.
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Dual Function of Phosphoubiquitin in Activation of Parkin [Molecular Bases of Disease]

June 9th, 2016 by Walinda, E., Morimoto, D., Sugase, K., Shirakawa, M.

Mutations in the gene encoding parkin, an auto-inhibited E3 ubiquitin ligase that functions in the clearance of damaged mitochondria, are the most common cause of autosomal recessive juvenile Parkinsonism. The mechanism regulating parkin activation remains poorly understood. Here we show, by using isothermal titration calorimetry, solution NMR and fluorescence spectroscopy, that parkin can bind ubiquitin and phosphomimetic ubiquitin by recognizing the canonical hydrophobic patch and C-terminus of ubiquitin. The affinity of parkin for both phosphomimetic and unmodified ubiquitin is markedly enhanced upon removal of the ubiquitin-like (UBL) domain of parkin. This suggests that the agonistic binding of ubiquitin to parkin in trans is counterbalanced by the antagonistic activity of the parkin UBL domain in cis. Intriguingly, UBL binding is enthalpy-driven, whereas ubiquitin binding is driven by an increase in the total entropy of the system. These thermodynamic differences are explained by different chemistry in the ubiquitin- and UBL- binding pockets of parkin and, as shown by molecular dynamics simulations, are not a consequence of changes in protein conformational entropy. Indeed, comparison of conformational fluctuations reveals that the RING1-IBR element becomes considerably more rigid upon complex formation. A model of parkin activation is proposed in which E2~Ub binding triggers large-scale diffusional motion of the RING2 domain toward the ubiquitin-stabilized RING1-IBR assembly to complete formation of the active parkin/E2~Ub transfer complex. Thus, ubiquitin plays a dual role in parkin activation by competing with the inhibitory UBL domain and stabilizing the active form of parkin.

Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: Insights on Leucine and Aliphatic Glucosinolate Biosynthesis [Protein Structure and Folding]

May 2nd, 2016 by Lee, S. G., Nwumeh, R., Jez, J. M.

Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different beta-hydroxyacids in the leucine and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg2+ or NAD+. These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the beta-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.
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Formation of a chloride-conducting state in the Maltose ATP-Binding Cassette (ABC) transporter [Protein Structure and Folding]

April 7th, 2016 by Carlson, M. L., Bao, H., Duong, F.

ABC transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations in order to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ABC transporter into a channel.

DNA Polymerase III, but not Polymerase IV, Must be Bound to {tau}-Containing DnaX Complex to Enable Exchange into Replication Forks [Enzymology]

April 7th, 2016 by Yuan, Q., Dohrmann, P. R., Sutton, M. D., McHenry, C. S.

Examples of dynamic polymerase exchange have been previously characterized in the model systems provided by coliphages T4 and T7. Using a dominant negative D403E Pol III α that can form initiation complexes and sequester primer termini, but not elongate, we investigated the possibility of exchange at the E. coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a containing DnaX complex, did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ, showed that τ1, τ2, or τ3 -DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size and gaps between fragments, possessed the ability to challenge pre-established replication forks and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.
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Structural Divergence of the Group I Intron-Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases that Function in RNA Splicing [RNA]

April 1st, 2016 by Lamech, L. T., Saoji, M., Paukstelis, P. J., Lambowitz, A. M.

The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mt tRNATyr and promote the splicing of autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon-binding domains and that the catalytic domain uses a newly evolved group I intron-binding surface, which includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA-binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined X-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the A. fumigatus mtTyrRS showed that the overall topology of the group I intron-binding surface is conserved, but with variations in key intron-binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions that also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron-specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.
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Role of glyoxylate shunt in oxidative stress response [Microbiology]

April 1st, 2016 by Ahn, S., Jung, J., Jang, I.-A., Madsen, E. L., Park, W.

The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the TCA cycle. The GS bypasses the carbon dioxide-producing steps of the TCA cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be upregulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including upregulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer: a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.

Entry of Bluetongue virus capsid requires the late endosomal specific lipid lysobisphosphatidic acid [Lipids]

April 1st, 2016 by Patel, A., Mohl, B.-P., Roy, P.

The entry of viruses into host cells is one of the key processes for the infection.. The mechanisms of cellular entry for enveloped virus have been well studied. The fusion proteins as well as the facilitating cellular lipid factors involved in the viral fusion entry process have been well characterized. The process of non-enveloped virus cell entry, in comparison, remains poorly defined, particularly for large complex capsid viruses of the family Reoviridae, which comprises a range of mammalian pathogens. These viruses enter cells without the aid of a limiting membrane and thus cannot fuse with host cell membranes to enter cells. Instead, these viruses are believed to penetrate membranes of the host cell during endocytosis. However, the molecular mechanism of this process is largely undefined. Here we show utilizing an in vitro liposome penetration assay and cell biology that Bluetongue virus (BTV), an archetypal member of the Reoviridae, utilizes the late endosomal specific lipid lysobisphosphatidic acid (LBPA) for productive membrane penetration and viral entry. Further we provide preliminary evidence that LBPA facilitates pore expansion during membrane penetration suggesting a mechanism for lipid factor requirement of BTV. This data indicates that despite the lack of a membrane envelope, the entry process of BTV is similar in specific lipid requirements to enveloped viruses that enter cells through the late endosome. These results are the first, to our knowledge, to demonstrate that a large non-enveloped virus of the Reoviridae has specific lipid requirements for membrane penetration and host cell entry.