The structure of the CstF-77 homodimer provides insights into CstF assembly

The cleavage stimulation factor (CstF) is essential for the first step of poly(A) tail formation at the 3' ends of mRNAs. This heterotrimeric complex is built around the 77-kDa protein bridging both CstF-64 and CstF-50 subunits. We have solved the crystal structure of the 77-kDa protein from Encephalitozoon cuniculi at a resolution of 2Å. The structure folds around 11 Half-a-TPR repeats defining two domains. The crystal structure reveals a tight homodimer exposing phylogenetically conserved areas for interaction with protein partners. Mapping experiments identify the C-terminal region of Rna14p, the yeast counterpart of CstF-77, as the docking domain for Rna15p, the yeast CstF-64 homologue.     

Chemical shift assignments of a minimal Rna14p/Rna15p heterodimer from the yeast cleavage factor IA complex

The two yeast proteins Rna14p and Rna15p form part of the cleavage/polyadenylation factor IA (CF IA) complex that is involved in the 3′ processing of pre-mRNA. Association of the two proteins is mediated by a small C-terminal peptide from Rna14p and a region in Rna15p that corresponds to the hinge domain first identified within the human orthologue. Here I report the ¹H, ¹³C and ¹⁵N spectral assignments for a bacterially co-expressed heterodimer of Rna14p/Rna15p. Further analysis of secondary chemical shifts reveals that both peptides are predominantly α-helical within the complex.     

Protein Hit1, a novel box C/D snoRNP assembly factor,controls cellular concentration of the scaffolding protein Rsa1 by direct interaction

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82–R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3′-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p_317–352–Hit1p_70–164 complex reveals a novel mode of protein–protein association explaining the strong stability of the Rsa1p–Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p–Rsa1p–Hit1p heterotrimer.     

Novel Protein-Protein Contacts Facilitate mRNA 3′-Processing Signal Recognition by Rna15 and Hrp1

Precise 3′-end processing of mRNA is essential for correct gene expression, yet in yeast, 3′-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3′-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3′-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.     

Apamin Does Not Inhibit Human Cardiac Na+ Current,L-type Ca2+ Current or Other Major K+ Currents

Background

Apamin is commonly used as a small-conductance Ca²⁺-activated K⁺ (SK) current inhibitor. However, the specificity of apamin in cardiac tissues remains unclear.

Objective

To test the hypothesis that apamin does not inhibit any major cardiac ion currents.

Methods

We studied human embryonic kidney (HEK) 293 cells that expressed human voltage-gated Na⁺, K⁺ and Ca²⁺ currents and isolated rabbit ventricular myocytes. Whole-cell patch clamp techniques were used to determine ionic current densities before and after apamin administration.

Results

Ca²⁺ currents (CACNA1c+CACNB2b) were not affected by apamin (500 nM) (data are presented as median [25^th percentile;75^th percentile] (from –16 [–20;–10] to –17 [–19;–13] pA/pF, P = NS), but were reduced by nifedipine to –1.6 [–3.2;–1.3] pA/pF (p = 0.008). Na⁺ currents (SCN5A) were not affected by apamin (from –261 [–282;–145] to –268 [–379;–132] pA/pF, P = NS), but were reduced by flecainide to –57 [–70;–47] pA/pF (p = 0.018). None of the major K⁺ currents (I _Ks, I _Kr, I _K1 and I _to) were inhibited by 500 nM of apamin (KCNQ1+KCNE1, from 28 [20]; [37] to 23 [18]; [32] pA/pF; KCNH2+KCNE2, from 28 [24]; [30] to 27 [24]; [29] pA/pF; KCNJ2, from –46 [–48;–40] to –46 [–51;–35] pA/pF; KCND3, from 608 [505;748] to 606 [454;684]). Apamin did not inhibit the I _Na or I _CaL in isolated rabbit ventricular myocytes (I _Na, from –67 [–75;–59] to –68 [–71;–59] pA/pF; I _CaL, from –16 [–17;–14] to –14 [–15;–13] pA/pF, P = NS for both).

Conclusions

Apamin does not inhibit human cardiac Na⁺ currents, L-type Ca²⁺ currents or other major K⁺ currents. These findings indicate that apamin is a specific SK current inhibitor in hearts as well as in other organs.     

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S_p)-adenosine cyclic 3′:5′-monophosphorothioate ((S_p)-cAMPS)) and the other a diastereoisomeric antagonist ((R_p)-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91–244)·C-subunit, by using fluorescence spectroscopy and amide H/²H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91–244), and the effects of cAMP, (S_p)-cAMPS, and (R_p)-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R_p)-cAMPS on amide exchange of the RIα(91–244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91–244). Surprisingly, (R_p)-cAMPS not only increased the affinity of RIα(91–244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R_p)-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R_p)-cAMPS-induced increase in affinity of RIα(91–244) toward the C-subunit indicates that (R_p)-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.Cyclic AMP-dependent protein kinase (PKA)¹ plays a crucial role in a plethora of cellular functions. All isoforms of PKA are composed of two catalytic (C) subunits and homodimeric regulatory (R) subunits (1–3). As the name implies, cAMP is a major PKA regulator (4). Much progress has been made in the last decade in delineating the molecular basis of action of cAMP. An important tactic in this endeavor has been through the comparison of the effects of cAMP with those of two phosphorothioate cAMP analogs: (S_p)-cAMPS (a cAMP mimic) and (R_p)-cAMPS (an antagonist and a diastereoisomer of (S_p)-cAMPS). Although the importance of geometry of the sulfur substitution is critical in determining the pharmacological properties of the two phosphorothioate cAMP analogs, the molecular basis for this behavior is not fully understood. To date, these comparisons have only been made using either wild-type or truncated mutants of the type Iα regulatory subunit (RIα) that are free in solution, not complexed to the C-subunit. X-ray spectroscopic examination of ligand-bound RIα(92–379) complexes reveals few differences between ligand-bound complexes, but the (R_p)-cAMPS complex is structurally “looser” with higher thermal factors than complexes formed with either cAMP or (S_p)-cAMPS (5). This is consistent with the observation that both cAMP and (S_p)-cAMPS, but not (R_p)-cAMPS, raise the urea concentration required for wild-type RIα unfolding (6). Further insight into the structural basis of cAMP action stems from NMR spectroscopic comparison of the effects of (R_p)-cAMPS, cAMP, and (S_p)-cAMPS on chemical shifts and ¹⁵N relaxation of the RIα(119–244) mutant (7). In addition to producing fewer significant chemical shift changes than either cAMP or (S_p)-cAMPS, (R_p)-cAMPS binding is associated with enhanced millisecond to microsecond time scale backbone motions of a β-turn (β2,3 loop) and around the phosphate-binding cassette (PBC) (7).Further insight into the molecular basis of actions of cAMP and its analogs should come from the analysis of ligand-bound R·C complexes. Unfortunately, the large size of even the heterodimeric R·C complex (∼95 kDa) and the difficulty of preparing (R_p)-cAMPS·R·C-subunit crystals currently preclude the use of both NMR spectroscopy and x-ray crystallography. Consequently, we took two alternative lower resolution approaches to this issue. One approach involves the use of site-directed labeling combined with fluorescence spectroscopy to examine both the effects of cAMP and its analogs on R-C subunit binding kinetics and on the conformational dynamics of RIα(91–244). RIα(91–244) includes the “A” cyclic nucleotide binding (CNB) domain, the pseudosubstrate, and linker domains and represents the minimal segments necessary for high affinity C-subunit binding (Fig. 1) (8). The other approach involves an examination of the effects of cAMP and its analogs on solvent exposure/conformational flexibility of RIα(91–244)·C-subunit complex using H/²H amide exchange measured with a combination of mass spectrometry (ESI-Q-TOF) and proteolytic fragmentation. In the first approach, fluorescein maleimide (FM) was conjugated to three cysteine substitution mutants with the substitution sites located near or within the pseudosubstrate sequence, the linker domain, or αC (R92C, T104C, and R239C, respectively) of RIα(91–244) (Fig. 1). The time-resolved fluorescence anisotropy results suggest that cAMP and (S_p)-cAMPS reduce the interaction of the RIα linker domain and αC with the two peripheral R-C interaction sites on the C-subunit (so-called Sites 2 and 3) without affecting the interaction of the pseudosubstrate sequence with the active site cleft (so-called Site 1). Because of limitations of the amide H/²H exchange experiments, only the effects of (R_p)-cAMPS on H/²H amide exchange in RIα(91–244)·C-subunit complex could be investigated. The results showed that (R_p)-cAMPS induces a relatively widespread increase in amide exchange, indicating limited unfolding and/or enhanced conformational flexibility that is propagated almost globally through the C-subunit and, at least, part of RIα. These conformational changes were accompanied by a 5-fold increase in the affinity of RIα(91–244) toward C-subunit, suggesting that, at least, some of the (R_p)-cAMPS effects are mediated by an increase in internal entropy. Finally, the (R_p)-cAMPS-induced increase in R-C affinity indicates that (R_p)-cAMPS is better described as an inverse agonist because the basal activity of the PKA holoenzyme should be decreased by (R_p)-cAMPS.Open in a separate windowFig. 1.Overview of PKA structure and cAMP analogs. A, domain organization of RIα showing the domain boundaries of RIα(91–244) where the pseudosubstrate in green is connected to CNB-A domain in blue by a linker segment. B, structure of RIα(91–244) in the C-subunit-bound conformation (Protein Data Bank code 1U7E (23)) showing the pseudosubstrate in green, linker in yellow, and helical subdomain comprising helices αN, αA, αB, and αC in blue and β-subdomain in tan. The PBC is in red. C, structure of the C·RIα(91–244) holoenzyme showing the C-subunit in tan and RIα(91–244) in blue. Sites for introduction of cysteines by site-directed mutagenesis are represented by red circles. The cAMP binding site (PBC) is in red. D, structure of cAMP showing the 2′-OH group and 3′–5′ phosphodiester bond. The exocyclic oxygens upon replacement with sulfur atoms to generate the (S_p)-cAMPS and (R_p)-cAMPS diastereomers are highlighted.     

Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 Å resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB–dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB–ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L₄₅ plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA–PriB–ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.     

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

O₂ sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1–F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1–cullin-1–F-box protein–Rbx1 subcomplex of E3^SCFUb ligases. E3^SCFUb ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O₂ availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA–Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O₂-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3^SCFUb ligases that regulate O₂-dependent developmental progression.Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a protein-by-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs)¹ are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1–FBP heterodimer (3–5). Deneddylation mediated by the eight-subunit COP9 signalosome is required for in vivo activity, suggesting that Cand1 serves as a substrate exchange factor to allow for re-equilibration of SCF complexes from preexisting subunits. Each reaction cycle requires the exchange of a new E2-Ub and typically assembles a K48-linked polyUb chain that is recognized by the proteasome. Substrate specificity is conferred by FBPs, a gene family that numbers 69 in humans, 20 in budding yeast, 300 in Caenorhabditis elegans, and ∼800 in Arabidopsis. Some characterized FBPs can recognize perhaps a dozen or more substrates, and the coding of recognition and the meaning of their control by the same FBP is under intense investigation (6). Recognition is often activated by posttranslational modification of the substrate (often phosphorylation). Regulation of SCF Ub ligases has centered on the neddylation cycle, which potentially influences all seven known CRLs. Regulation of Skp1, investigated in this paper, would be specific to CRLs possessing Skp1, which include CRL1 and possibly the minor class CRL7 (7).Open in a separate windowFig. 2.Skp1 modification pathway and global analysis of Skp1 interactions. A, Skp1 is sequentially modified by the indicated enzymes (in blue), resulting in the formation of a pentasaccharide at Pro143. B, model of the SCF complex in the context of the overall E3 Ub ligase, from studies in yeast, plants, and animals. Catalysis involves transfer of Ub from an exchangeable Ub-E2 conjugate to the substrate. Removal of Nedd8 by the COP9 signalosome facilitates binding of Cand1 to Cul1, which inhibits binding of Skp1 to Cul1. C, D, vegetative (growth stage) cells were filter-lysed, and a cytosolic fraction prepared via ultracentrifugation was chromatographed on a Superose 12 gel filtration column. Fractions were analyzed via Western blotting (representative examples are shown in C) followed by densitometry (D). The elution position of free Skp1 from a separate trial is indicated.The basic SCF model is thought to be widespread among eukaryotes but has been extensively studied only in fungi/yeasts, plants, and animals. The broad phylogeny represented by protists includes many benign and pathogenic unicellular organisms of great economic, health, and environmental impact. Emerging evidence reveals that Skp1 in some of these groups is subject to a novel form of prolyl 4(trans)-hydroxylation and complex glycosylation (8). The roles of these Skp1 modifications have been most studied in the social amoeba Dictyostelium, which undergoes a starvation-induced developmental program during which individual amoebae chemotactically aggregate into an initial mound that then elongates into a migratory slug. Under appropriate conditions, the slug reorganizes to form a fruiting body consisting of a ball of spores supported by a vertical cellular stalk. The slug-to-fruit switch, referred to as culmination, and sporulation are regulated by checkpoints that are sensitive to multiple factors, including O₂ (9–11). Functional studies of Dictyostelium Skp1 hydroxylation and glycosylation reveal roles in regulating the O₂ dependence of culmination and sporulation (12–14). For example, wild-type (wt) cells require 7% to 10% O₂ and phyA^– requires 18% to 21% O₂ in order to achieve 50% spore formation (a quantitative measure of fruiting body formation), whereas glycosylation mutants exhibit a complex pattern of intermediate requirements (13). In addition, at 21% O₂, phyA^– cells require an additional 3 to 4 h to complete development relative to their wt counterparts (14). In the apicomplexan Toxoplasma gondii, PhyA is also required for Skp1 glycosylation, and phyA^– parasites are deficient in proliferation, especially at low O₂ (15).The idea that O₂ availability is rate limiting for Skp1 modification was originally based on the observation that the Dictyostelium phyA^– phenotype mimics that of wt cells in low O₂ (9). However, the majority of Skp1 is hydroxylated and glycosylated in wt cells even at low O₂ levels where culmination is blocked or delayed. Further analysis of a submerged development model, in which terminal development depended on an atmosphere of 70% to 100% O₂ in order to overcome the diffusion barrier posed by the water layer, showed that at atmospheric O₂ levels of 5% to 21% where sporulation was blocked, unmodified Skp1 accumulated to a higher level than at permissive O₂ levels (10). As Skp1 modifications are thought to be irreversible, this likely resulted from slow hydroxylation of newly synthesized Skp1. To address this in a more physiological setting, we investigated nascent Skp1 directly using metabolic labeling with [³⁵S]Met/Cys and verified that the rate of hydroxylation of newly synthesized Skp1 polypeptide was indeed inversely proportional to O₂ levels, which makes PhyA-mediated hydroxylation of Skp1 an excellent candidate for the primary O₂ sensor for culmination.These modifications of Skp1 are of interest as a novel mechanism regulating the SCF ligase. Previously, we showed that hydroxylation and glycosylation of Dictyostelium Skp1 affect its conformation and promote binding to a soluble FBP, guinea pig Fbs1, in studies of purified proteins (16). Here we show that Dictyostelium Skp1 is indeed a subunit of a canonical SCF complex, as expected. The significance of undermodified Skp1 was examined via interactome analysis of Skp1 isoforms that accumulate in modification pathway mutants. Our findings revealed a lower abundance of SCF complexes than in wt cells, suggesting that Skp1 modification may promote SCF assembly and E3^SCFUb ligase activities that control timely turnover of select proteins involved in developmental progression.     

Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago–Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago–Y14. Here we show that PYM is a cytoplasmic RNA-binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago–Y14 by means of its N-terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 Å resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense-mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD.     

Probing the Conformation of the Fibronectin III1�C2 Domain by Fluorescence Resonance Energy Transfer

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III₁ and III₂, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III_1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III_1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III_1–2.Fibronectin (FN)³ is a 500-kDa modular dimeric protein and a major component of the extracellular matrix. It exists in the blood and other body fluids as a soluble compact molecule and undergoes cell-mediated assembly to form an insoluble three-dimensional fibrillar matrix (reviewed in Ref. 1). The process of FN matrix assembly has been implicated in embryonic development, wound healing, and cancer (2–4). FN is composed of type I–III modules, and sets of these modules comprise binding domains for cells and for other extracellular matrix components (see Fig. 1A). Three of these binding domains are essential for matrix assembly (1). Integrin receptor interactions with the cell-binding domain tether disulfide-bonded FN dimers to the cell surface, where FN-FN interactions involving the N-terminal assembly domain form dimers into fibrils. In addition to these essential domains, other FN-binding sites have been implicated in assembly. In particular, the III_1–2 FN-binding domain plays a regulatory role in matrix assembly. Within this domain reside a cryptic FN-binding site in III₁ and a site available for FN binding in the native form of III₂ (5–8). Recombinant FN lacking III₁ is assembled into a matrix at wild-type levels, but that lacking the III_1–2 domain results in short immature FN fibrils (8). Peptides derived from the III_1–2 domain or antibodies against III_1–2 block matrix assembly by cultured cells (9–11). Furthermore, FN binding to this region is enhanced when FN is mechanically stretched (12). Taken together, these results suggest that conformational changes in the III_1–2 domain may control its interactions during FN assembly.Open in a separate windowFIGURE 1.The FN III_1–2 FRET conformational sensor. A, representation of the domain structure of FN and major interaction sites. FN is composed of repeating modules that form binding domains for other FN molecules, cell receptors, and other extracellular matrix components as indicated. The first two type III modules III₁ and III₂ (black), have FN-binding sites and regulate FN matrix assembly. The N-terminal 70-kDa region contains a matrix assembly domain with FN-binding activity. The cell-binding domain (cell), the heparin-binding domain (heparin), the dimerization site (SS), and the alternatively spliced type IIIA (A), IIIB (B), and variable regions (V) are indicated. 70kD, N-terminal 70-kDa FN fragment. B, schematic of proposed model of III_1–2 domain conformation. Panel i, in solution, the FN-binding sites in III₁ and III₂ (hatched areas) are sequestered through domain orientations that are facilitated by the linker between modules (thin line). Panel ii, binding sites are exposed through conformational changes resulting from cell-mediated extension of FN (arrows). The length of the linker and the height and width of the modules are drawn to scale for a linear peptide and published data on FN type III modules, respectively. C, ribbon diagram representation of CIIIY, a FRET sensor of the model in B (panel i), oriented with N and C termini 50 Å apart. CIIIY consists of the III_1–2 domain with CFP at the N terminus and YFP at the C terminus.To more fully understand the roles of native and cryptic FN-binding sites in matrix assembly, the conformational dynamics of III_1–2 must be characterized. One approach to this problem is to tag III_1–2 with fluorescent probes, which, in conjunction with fluorescent resonance energy transfer (FRET), create a molecular conformational sensor. FRET involves the radiationless transfer of energy from an excited donor fluorophore to an acceptor fluorophore, a process that is very sensitive to the distance between the two fluorophores (13–15). Two fluorescent protein variants, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), are highly related to green fluorescent protein (GFP). Because the emission spectrum of CFP is well matched to the excitation spectrum of YFP, these two fluorophores have been widely used as a donor-acceptor pair in FRET studies (13–15).In this study, we describe a FRET conformational sensor designed to test the idea that intramolecular interactions between III₁ and III₂ sequester key FN-binding and assembly sites. We show that III_1–2 with CFP and YFP fused to the N and C termini, respectively, displays a clear FRET signal, indicating that the attached fluorescent proteins and thus the ends of III_1–2 are in close proximity. FRET data from III_1–2 mutants support the presence of a stabilizing intermodule salt bridge that regulates FN-binding activity.     

A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a φ29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.The DNA packaging motor of double-stranded DNA bacteriophages translocates genomic DNA into a preformed procapsid to near crystalline density and is the strongest motor characterized to date. The packaging motor of the Bacillus subtilis phage φ29 can work against 57 piconewtons of internal force and translocate 2 bp of DNA per ATP hydrolyzed at a maximum velocity of 103 bp/s (1, 2). The motor complex is assembled on a dodecamer of the connector protein, which replaces a pentameric vertex in the procapsid and serves both as a portal for DNA passage and the docking site for the other packaging components (3).To successfully package a full-length genome, incorporation of one and only one connector vertex is essential (4). In vivo, nearly every assembled procapsid has one and only one connector vertex and is able to package DNA and mature into an infectious phage (5). This narrow distribution in which 95% of particles have a single connector vertex cannot be explained by random statistical incorporation. The control mechanism is coupled to the procapsid assembly process. Procapsid assembly requires the copolymerization of hundreds of copies each of the capsid and scaffolding proteins as well as a dodecamer of the portal or connector protein. The scaffolding protein acts to both activate the coat protein for assembly and ensure proper form determination. In the absence of scaffolding protein, uncontrolled polymerization results in the assembly of aberrant structures. In a properly assembled procapsid, the portal protein is located at one vertex, whereas scaffolding protein occupies the bulk of the interior space and is subsequently removed during DNA packaging by either proteolysis or simple release. Mutational studies have indicated that scaffolding protein is involved either directly or indirectly in the incorporation of the connector vertex during procapsid assembly in a variety of phages (6–8).In φ29, the connector vertex is specifically incorporated at one of the two 5-fold vertices lying on the long axis of a prolate procapsid composed of 235 copies of capsid protein and containing ∼180 copies of scaffolding protein (9, 10). The structure of the 33-kDa connector protein subunit consists of three long central α-helices bridging wide and narrow domains that are rich in β-sheets and extended polypeptides (Fig. 1A) (10–12). The 12 subunits are arranged to form a 75-Å-long tapered grommet-shaped structure with an external diameter of 69 Å at the wide end and 33 Å at the narrow end. By fitting the crystal structure of the connector dodecamer into the cryo-EM¹ density of the procapsid, the orientation of connector at the unique vertex of the procapsid was revealed. The wide domain of connector protein lies inside the procapsid, and the narrow domain is exposed to the exterior and makes contacts with the other parts of the motor complex (11). The 11-kDa scaffolding protein subunits form nanomolar affinity homodimers resembling arrows in solution. Each subunit contributes one side of the arrowhead and one-half of the long coiled coil shaft (Fig. 1B) (13). The subunit structure consists of three helical segments. A three-turn N-terminal helix (α1) followed by a five-residue loop, and an antiparallel five-turn helix (α2) makes up the arrowhead and part of the proximal part of the shaft. A three-residue loop and a seven-turn helix (α3) complete the shaft. The C-terminal 15 residues, which interact with capsid protein as determined in the in vitro assembly assay, are disordered in the crystal structure (14).Open in a separate windowFig. 1.The x-ray crystal structures of connector protein (Protein Data Bank code 1FOU, chains A and B) (A) and scaffolding protein (Protein Data Bank code 1NO4, chains A and B) (B).We have recently reported the development of an in vitro assembly system for phage φ29 in which purified connector protein complex can be successfully incorporated (15). The addition of connector protein dodecamers to coat and scaffolding subunits accelerated the rate of assembly and lowered the critical concentration, suggesting involvement in nucleation of assembly (15). Here we used native mass spectrometry, chemical cross-linking, and mutational analysis to characterize the interactions between the connector and the scaffolding proteins and develop a model of the scaffolding-connector complex, which provides a molecular model of how scaffolding protein might mediate stringent incorporation of one and only one connector dodecamer.     

The unstructured C-terminus of the tau subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the alpha subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τ_C16. We show that the extreme C-terminal region of τ_C16 constitutes the site of interaction with α. The τ_C16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τ_C16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (K_D) of the α−τ_C16 complex to be ~260pM. Competition with immobilized τ_C16 by τ_C16 derivatives for binding to α gave values of K_D of 7μM for the α−τ_C16Δ7 complex. Low-level expression of the genes encoding τ_C16 and τ_C167, but not τ_C16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τ_C16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τ_C24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τ_C16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ.     

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Glycogen synthase kinase-3β (GSK3β) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia–reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia–reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3β might have a role in the Ca²⁺ transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3β protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3β specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP₃Rs) Ca²⁺ channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3β decreased protein interaction of IP₃R with the Ca²⁺ channeling complex, impaired SR/ER Ca²⁺ release and reduced the histamine-stimulated Ca²⁺ exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3β activity and IP₃R phosphorylation, which leads to enhanced transfer of Ca²⁺ from SR/ER to mitochondria. Inhibition of GSK3β at reperfusion reduced both IP₃R phosphorylation and SR/ER Ca²⁺ release, which consequently diminished both cytosolic and mitochondrial Ca²⁺ concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3β at reperfusion diminishes Ca²⁺ leak from IP₃R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca²⁺ overload and subsequent cell death.Glycogen synthase kinase-3 (GSK3) was originally identified as a phosphorylating kinase for glycogen synthase.^{1, 2} It has two isoforms, α and β, that possess strong homology in their kinase domains with, however, distinct functions.³ GSK3 is constitutively active but it can be inhibited by phosphorylation on serine 21 (Ser21) for GSK3α and Ser9 for GSK3β.⁴ In the heart, GSK3β has several important roles in cardiac hypertrophy⁵ and ischemia–reperfusion (IR) injury.⁶ Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening.^{6, 7, 8, 9} The mechanism by which GSK3β delays PTP opening still remains unclear. It has been reported that GSK3β could interact with ANT at the inner mitochondrial membrane in the heart⁹ and/or to phosphorylate voltage-dependent anion channel (VDAC) and cyclophilin D (CypD) in cancer cells.^{10, 11} GSK3β also has other proposed mechanisms of action, including a poorly characterized role in calcium (Ca²⁺) homeostasis regulation¹² and protein–protein interactions,⁹ as well as functions in different subcellular fractions such as the nucleus, cytosol and mitochondria.¹³Reperfusion is the most powerful intervention to salvage ischemic myocardium. However, it can also paradoxically lead to cardiomyocyte injury and death.¹⁴ One of the main actors of this lethal reperfusion injury is cellular Ca²⁺ overload,¹⁵ which results in part from excessive sarco/endoplasmic reticulum (SR/ER) Ca²⁺ release and Ca²⁺ influx through the plasma membrane (e.g. through L-type Ca²⁺channel and NCX (sodium-calcium exchanger)).¹⁶ Although ryanodine receptors (RyRs) are the major cardiac SR/ER Ca²⁺-release channels involved in excitation–contraction coupling (ECC)¹⁷ and ischemia–reperfusion (IR) injury,¹⁸ recent studies reported an increasing role for inositol 1,4,5-trisphosphate receptors (IP₃Rs) Ca²⁺-release channels in the modulation of ECC and cell death.^{19, 20} Ca²⁺-handling proteins of ER and mitochondria are highly concentrated at mitochondria-associated ER membranes (MAMs), providing a direct and proper mitochondrial Ca²⁺ signaling, including VDAC, Grp75 and IP₃R1.^{20, 21, 22}Here, we provide evidence that, following IR, a fraction of cellular GSK3β is localized at the SR/ER and MAMs. At the MAMs interface, GSK3β can specifically interact and regulate the protein composition of the IP₃R Ca²⁺ channeling complex and modulate Ca²⁺ transfer between SR/ER and mitochondria. These findings support a novel mechanism of action of GSK3β in cell death process during reperfusion injury.     

A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica

Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix–turn–helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix α1 contacts tRNA whereas helix α2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.