Pat1 contributes to the RNA binding activity of the Lsm1-7–Pat1 complex

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5′-to-3′ exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7–Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7–Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7–Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7–Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7–Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7–Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.     

High affinity binding of the transcobalamin II–cobalamin complex and mRNA expression of haptocorrin by human mammary epithelial cells

Little is known about the acquisition of cobalamin by the mammary gland and its secretion into milk. Human milk and plasma contain at least two types of cobalamin binding proteins: transcobalamin II (TC) and haptocorrin (HC). In plasma, TC is responsible for the transport of cobalamin to tissues and cells; however, cobalamin in milk is present exclusively bound to HC. We show that human mammary epithelial cells (HMEC) exhibit high affinity for TC; Scatchard analysis revealed a single class of binding sites for the TC–[⁵⁷Co]cyanocobalamin complex with a dissociation constant (K_d) of 4.9×10⁻¹¹ M. Uptake of the TC–[⁵⁷Co]cyanocobalamin complex at 37°C was saturable by 24 h. Binding of free [⁵⁷Co]cyanocobalamin to HMEC was not saturable and very limited binding of the HC–[⁵⁷Co]cyanocobalamin complex was observed. Expression of the haptocorrin gene by HMEC was confirmed by Northern blot and PCR analysis. Thus, a specific cell surface receptor for the TC–cobalamin complex exists in the mammary gland and once cobalamin is internalized, it may be transferred to HC and subsequently secreted into milk as a HC–cobalamin complex.     

Activation of brain hexokinase by magnesium ions and by magnesium ion–adenosine triphosphate complex

1. An alternative explanation for the kinetic data obtained by Bachelard (1971) for the brain hexokinase reaction is presented. 2. Apparently sigmoidal saturation curves for MgATP²⁻ based upon Bachelard's (1971) studies can be corrected to hyperbolic curves by use of a stability constant for MgATP²⁻ complex formation. 3. A number of other effects related to the concentration-dependent stability of the MgATP²⁻ complex and to the presence of the inhibitory free uncomplexed ATP⁴⁻ concentration are also explained in terms of a non-allosteric role for either Mg²⁺ or MgATP²⁻ fully consistent with a number of previous reports on this enzyme. 4. A brief discussion of the validity of Hill plots in studies of multisubstrate co-operative enzymes is presented. 5. A simple model is presented that demonstrates how enzymes obeying Michaelis–Menten kinetics can demonstrate sigmoidal velocity responses if the true substrate of the reaction is the metal–substrate complex.     

The fine-tuning of TRAF2–GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K_d=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K_d≥2.6 μM) or is inactivated by oxidation (K_d=1.7 μM). This indicates that GSTP1-1''s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex''s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1''s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex''s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH''s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.     

Haptoglobin-hemoglobin binding involves the heavy chain of haptoglobin and the α and β chains of hemoglobin

Previous studies from this laboratory have demonstrated unambiguously that the isolated β chain of human adult hemoglobin binds human haptoglobin (Hp). In the present work, the ability of the isolated subunits of haptoglobin and hemoglobin to form complexes is investigated. In quantitative radiometric adsorbent titrations, the H chain of haptoglobin bound to hemoglobin whereas the L chain had no binding activity. Also, the H chain of haptoglobin bound to the isolated α and β subunits of hemoglobin, but its binding to the α or β chain was less than the binding it exhibits to hemoglobin. The isolated L chain was able to reassociate with the H chain to form a complex that binds to hemoglobin or its subunits. Although the L chains had no binding activity, its association with the H chain increased the binding of the latter to Hb or its isolated α and β subunits suggesting a more indirect role for the L chain in haptoglobin-hemoglobin interactions.     

Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.     

Mapping of the binding sites involved in PSP94–CRISP-3 interaction by molecular dissection of the complex

Background

Human Prostate Secretory Protein of 94 amino acids (PSP94) has been shown to bind human CRISP-3 (cysteine-rich secretory protein 3) with very high affinity. CRISP-3 belongs to the CRISP family of proteins having a PR-1 (pathogenesis related protein 1) domain at its N-terminal and ion channel regulatory (ICR) domain at its C-terminal connected by a hinge region. Functional significance of this complex is not yet known.

Methods

In order to identify the residues and/or regions involved in PSP94–CRISP-3 interaction, site-directed mutagenesis was employed. Effect of the mutations on the interaction was studied by co-immunoprecipitation (Co-IP).

Results

For PSP94, amino acids Y³, F⁴, P⁵⁶ and the C-terminal β-strand were found to be crucial for interacting with CRISP-3. A disulfide bond between the two domains of PSP94 (C³⁷A–C⁷³A) was also important for this interaction. In case of CRISP-3, the N-terminal domain alone could not maintain a strong interaction with PSP94 but it required presence of the hinge region and not the C-terminal domain. Apart from CRISP-3, CRISP-2 was also found to interact with human PSP94. Based on our findings the most likely model of PSP94–CRISP-3 complex has been proposed.

Conclusion

The terminal β-strands of PSP94 contact the first α-helix and the hinge region of CRISP-3.

General significance

Involvement of the hinge region of CRISPs in interaction with PSP94 may affect the domain movement of CRISPs essential for the ion-channel regulatory activity resulting in inhibition of this activity.     

Structure of a protein–detergent complex: the balance between detergent cohesion and binding

Despite the major interest in membrane proteins at functional, genomic, and therapeutic levels, their biochemical and structural study remains challenging, as they require, among other things, solubilization in detergent micelles. The complexity of this task derives from the dependence of membrane protein structure on their anisotropic environment, influenced by a delicate balance between many different physicochemical properties. To study such properties in a small protein–detergent complex, we used fluorescence measurements and molecular dynamics (MD) simulations on the transmembrane part of glycophorin A (GpAtm) solubilized in micelles of dihexanoylphosphatidylcholine (DHPC) detergent. Fluorescence measurements show that DHPC has limited ability to solubilize the peptide, while MD provides a possible molecular explanation for this. We observe that the detergent molecules are balanced between two different types of interactions: cohesive interactions between detergent molecules that hold the micelle together, and adhesive interactions with the peptide. While the cohesive interactions are detergent mediated, the adhesion to the peptide depends on the specific interactions between the hydrophobic parts of the detergent and the topography of the peptide dictated by the amino acids. The balance between these two parameters results in a certain frustration of the system and rather slow equilibration. These observations suggest how molecular properties of detergents could influence membrane protein stabilization and solubilization.     

Modelling and predicting the binding mechanics of HIV P1053-0.5β antibody complex

Simulating antigen–antibody interactions is crucial in understanding the mechanics of antigen–antibody binding in medical science. In this study, molecular dynamics simulations are performed to analyse the dissociation of the P1053-0.5β antibody complex structure. The two-dimensional free energy profiles of the complex structure are extracted using the weighted histogram analysis method, and the binding pathway is then predicted using a modified form of the MaxFlux-PRM method. The simulation results suggest that 10 amino residues (i.e. Leu11, Val13, Asp34, Arg112, Thr101, Gly127, Val229, Ser231, Ile235 and Arg236) play a key role in relaxing the antibody structure, thereby facilitating the binding of the 0.5β antibody-P1053 peptide system.     

Differential regulation of the calpain–calpastatin complex by the L-domain of calpastatin

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca^2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.     

Jasmonate induces biosynthesis of anthocyanin and proanthocyanidin in apple by mediating the JAZ1–TRB1–MYB9 complex

Jasmonate (JA) induces the biosynthesis of anthocyanin and proanthocyanidin. MdMYB9 is essential for modulating the accumulation of both anthocyanin and proanthocyanidin in apple, but the molecular mechanism for induction of anthocyanin and proanthocyanidin biosynthesis by JA is unclear. In this study, we discovered an apple telomere-binding protein (MdTRB1) to be the interacting protein of MdMYB9. A series of biological assays showed that MdTRB1 acted as a positive modulator of anthocyanin and proanthocyanidin accumulation, and is dependent on MdMYB9. MdTRB1 interacted with MdMYB9 and enhanced the activation activity of MdMYB9 to its downstream genes. In addition, we found that the JA signaling repressor MdJAZ1 interacted with MdTRB1 and interfered with the interaction between MdTRB1 and MdMYB9, therefore negatively modulating MdTRB1-promoted biosynthesis of anthocyanin and proanthocyanidin. These results show that the JAZ1–TRB1–MYB9 module dynamically modulates JA-mediated accumulation of anthocyanin and proanthocyanidin. Taken together, our data further expand the functional study of TRB1 and provide insights for further studies of the modulation of anthocyanin and proanthocyanidin biosynthesis by JA.     

Interpreting the structural mechanism of action for MT7 and human muscarinic acetylcholine receptor 1 complex by modeling protein–protein interaction

MT7 is a selective human muscarinic acetylcholine receptor 1 (hM1) allosteric binder with subnanomolar affinity. Understanding the binding mode of hM1–MT7 will give insights to discover small molecular ligand for hM1. MT7 is a peptide, and hM1 is a G-protein-coupled membrane receptor. Therefore, we have employed homology modeling, protein–protein docking, explicit membrane molecular dynamics (MD) simulations, and molecular mechanic/Poisson-Boltzmann surface area energy decomposition analysis approaches to reveal the hM1–MT7 binding mode. The binding mode is consistent with the experimental data. We have discovered that the binding mode consists of three interaction regions in five residue interaction clusters. By analyzing the cluster representative structures, the cluster residues form an interaction network, which shows a multiple-point-to-site binding mode. Hydrogen binding statistical analysis reveals that E170 (hM1) and R34 (MT7) are both locked in electrostatic cages with counter charges, respectively. This is confirmed by the dynamic distances calculation between these residues, and biological mutant experiments.     

Trafficking of the Menkes copper transporter ATP7A is regulated by clathrin-, AP-2–, AP-1–, and Rab22-dependent steps

The transporter ATP7A mediates systemic copper absorption and provides cuproenzymes in the trans-Golgi network (TGN) with copper. To regulate metal homeostasis, ATP7A constitutively cycles between the TGN and plasma membrane (PM). ATP7A trafficking to the PM is elevated in response to increased copper load and is reversed when copper concentrations are lowered. Molecular mechanisms underlying this trafficking are poorly understood. We assess the role of clathrin, adaptor complexes, lipid rafts, and Rab22a in an attempt to decipher the regulatory proteins involved in ATP7A cycling. While RNA interference (RNAi)–mediated depletion of caveolin 1/2 or flotillin had no effect on ATP7A localization, clathrin heavy chain depletion or expression of AP180 dominant-negative mutant not only disrupted clathrin-regulated pathways, but also blocked PM-to-TGN internalization of ATP7A. Depletion of the μ subunits of either adaptor protein-2 (AP-2) or AP-1 using RNAi further provides evidence that both clathrin adaptors are important for trafficking of ATP7A from the PM to the TGN. Expression of the GTP-locked Rab22a_Q64L mutant caused fragmentation of TGN membrane domains enriched for ATP7A. These appear to be a subdomain of the mammalian TGN, showing only partial overlap with the TGN marker golgin-97. Of importance, ATP7A remained in the Rab22a_Q64L-generated structures after copper treatment and washout, suggesting that forward trafficking out of this compartment was blocked. This study provides evidence that multiple membrane-associated factors, including clathrin, AP-2, AP-1, and Rab22, are regulators of ATP7A trafficking.     

Preferential activation by galanin 1–15 fragment of the GalR1 protomer of a GalR1–GalR2 heteroreceptor complex

The three cloned galanin receptors show a higher affinity for galanin than for galanin N-terminal fragments. Galanin fragment (1–15) binding sites were discovered in the rat Central Nervous System, especially in dorsal hippocampus, indicating a relevant role of galanin fragments in central galanin communication. The hypothesis was introduced that these N-terminal galanin fragment preferring sites are formed through the formation of GalR1–GalR2 heteromers which may play a significant role in mediating galanin fragment (1–15) signaling. In HEK293T cells evidence for the existence of GalR1–GalR2 heteroreceptor complexes were obtained with proximity ligation and BRET² assays. PLA positive blobs representing GalR1–GalR2 heteroreceptor complexes were also observed in the raphe-hippocampal system. In CRE luciferase reporter gene assays, galanin (1–15) was more potent than galanin (1–29) in inhibiting the forskolin-induced increase of luciferase activity in GalR1–GalR2 transfected cells. The inhibition of CREB by 50 nM of galanin (1–15) and of galanin (1–29) was fully counteracted by the non-selective galanin antagonist M35 and the selective GalR2 antagonist M871. These results suggested that the orthosteric agonist binding site of GalR1 protomer may have an increased affinity for the galanin (1–15) vs galanin (1–29) which can lead to its demonstrated increase in potency to inhibit CREB vs galanin (1–29). In contrast, in NFAT reporter gene assays galanin (1–29) shows a higher efficacy than galanin (1–15) in increasing Gq/11 mediated signaling over the GalR2 of these heteroreceptor complexes. This disbalance in the signaling of the GalR1–GalR2 heteroreceptor complexes induced by galanin (1–15) may contribute to depression-like actions since GalR1 agonists produce such effects.     

Backbone and sidechain methyl Ile (δ1), Leu and Val resonance assignments of the catalytic domain of the yeast mRNA decapping enzyme, Dcp2

Eukaryotic mRNA decapping by Dcp2 is the penultimate step in several mRNA decay pathways. To understand regulation of Dcp2 by ligand interactions, we have assigned the backbone and sidechain methyl Ile (δ1), Leu and Val chemical shifts of the catalytic domain of the S. Cerevisiae enzyme.     

Integration of non-vesicular and vesicular transport processes at the Golgi complex by the PKD–CERT network

Non-vesicular transport of ceramide from endoplasmic reticulum to Golgi membranes is essential for cellular lipid homeostasis. Protein kinase D (PKD) is a serine–threonine kinase that controls vesicle fission at Golgi membranes. Here we highlight the intimate connections between non-vesicular and vesicular transport at the level of the Golgi complex, and suggest that PKD and its substrate CERT, the ceramide transfer protein, play central roles in coordinating these processes by fine-tuning the local membrane lipid composition to maintain Golgi secretory function. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.