Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds RNA with low-nanomolar K d values and long half-lives of complexes (>100 min). This cannot be reconciled with the 1-2-min response time of regulation in vivo. We show that RNAs displace each other on Hfq on a short time scale by RNA concentrationdriven (active) cycling. Already at submicromolar concentrations of competitor RNA, half-lives of RNA-Hfq complexes are »1 min. We propose that competitor RNA associates transiently with RNA-Hfq complexes, RNAs exchange binding sites, and one of the RNAs eventually dissociates. This solves the ''strong binding-high turnover'' paradox and permits efficient use of the Hfq pool. The homohexameric Hfq ring displays two faces: proximal and distal. Hfq-RNA interactions show a preference of U-rich for proximal and A-rich RNA sequences for distal face binding (de Haseth and Uhlenbeck 1980a;Mikulecky et al. 2004). Simultaneous binding may occur on both sides as well, which could facilitate intermolecular base-pairing and regulation (Rajkowitsch and Schroeder 2007).Structures of Hfq from E. coli, Staphylococcus aureus, and Pseudomonas aeroginosa have been determined by X-ray crystallography (Schumacher et al. 2002;Sauter et al. 2003;Nikulin et al. 2005). Two cocrystal structures support two distinct binding surfaces: In S. aureus Hfq, AU 5 G RNA is bound around the inner rim of the proximal face (Schumacher et al. 2002), and E. coli Hfq has oligo-A bound on the distal face (Link et al. 2009 Holmqvist et al. 2010). Thus, if binding-competent RNAs were in molar excess, almost all Hfq would be bound to RNAs. Hfq-RNA dissociation rate constants in vitro are too low to be compatible with a biologically relevant time scale; half-lives of complexes are in the range of a generation time. If newly induced sRNAs only could access free Hfq after its dissociation from bound RNAs, their activity should be severely delayed. Yet, the time frame from induction of an sRNA to a significant regulatory effect is short (1-2 min) (Massé et al. 2003), and hence sRNAs can acquire Hfq rapidly. This highlights a paradox, with Hfq being tightly sequestered by the intracellular pool of RNAs, contrasted by the need of new sRNAs to rapidly access Hfq. We considered here a conventional cycling model (dissociative/passive) (Fig. 1A) and associative/active cycling (Fig. 1B). In model A, newly synthesized RNA (Fig. 1A, in red) can only bind Hfq after the resident RNA (Fig. 1A, in blue) has dissociated; i.e., the rate of binding of the incoming RNA is limited by the Hfq-RNA dissociation rate constant and is not affected by the concentration of the free RNA. In model B, free RNA transiently binds the Hfq-RNA complex, whereupon one of the RNAs eventually dissociates. Thus, the dissociation rate of the bound RNA is a function of the concentration of the free RNA (Fig. 1B). This would render cycling much more rapidly, and the intracellular pool of binder RNAs would rapidly equilibrate on Hfq. The two models are distinguishable, since th...

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.

We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.intrabodies | X-ray crystallography

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3′-diamino-4,4′-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating β-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 mol/(min⅐mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RI ␣ ) of cAMP-dependent protein kinase (K D ‫؍‬ 10 nM) and the heat-stable protein kinase inhibitor (K D ‫؍‬ 15 nM), but not with the type II regulatory subunit (RII ␣ , K D ‫؍‬ 2.3 M) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RI ␣ subunit and by protein kinase inhibitor in vitro, but only weakly by the RII ␣ subunit. The inhibition by the RI ␣ subunit is reversed by addition of nanomolar concentrations of cAMP (K a ‫؍‬ 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the C ␣ subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RII ␣ subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RI ␣ subunit and by phosphorylating the RII ␣ subunit in the absence of cAMP.