Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.

The signal recognition particle (SRP) and its receptor comprise a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolution. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP•SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers where important questions remain to be addressed.

SUMMARY The modular SCF ubiquitin ligases feature a large family of substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCFFbxw7 is extraordinarily stable but, in the Nedd8-deconjugated state, is rapidly disassembled by the cullin-binding protein Cand1. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1–Rbx1 equilibrates with multiple F-box Protein–Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F-box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh•4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh•FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, [1640][1641][1642][1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh•FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh•FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.

The pathway by which ubiquitin chains are generated on substrate via a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCF Cdc4 and SCF β-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.Attachment of a polyubiquitin chain with at least four ubiquitins linked together through their lysine 48 residue (Lys48) targets proteins to the proteasome for degradation.1 A cascade of three enzymes carries out the synthesis of polyubiquitin chains: a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2), and a ubiquitin ligase (E3).2 RING (really interesting new gene) E3s catalyze the direct transfer of ubiquitin from an E2 to a lysine on a target protein.3 SCF Cdc4 is the founding member of the largest family of E3s -the cullin-RING ubiquitin ligases (CRLs) that may comprise the majority of all human ubiquitin ligases.3 Thus, unraveling the mechanism of SCF will have broad functional ramifications for the preponderance of human E3s.Different pathways for ubiquitin chain assembly by RING E3s have been envisioned based on indirect evidence. On the one hand, Cdc34-SCF ubiquitylates substrates bearing a single Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms 3 Corresponding author. deshaies@caltech.edu. * These authors contributed equally to this work. Author ContributionsN.W.P. performed all computational modeling and experiments except G.K. performed the mass spec experiments in Fig. 1g. N.W.P., R.J.D., and S.O.S. conceived the experiments. N.W.P. and R.J.D. wrote the manuscript with editorial input from the other authors. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript ubiquitin significantly faster than non-ubiquitylated substrates,4,5 suggesting that it processively builds polyubiquitin chains on substrates with an initial slow transfer of ubiquitin followed by rapid elongation into a Lys48-linked polyubiquitin chain. On the other hand, the E2 Ube2g2, a close relative of Cdc34, collaborates with the E3 gp78 to build a polyubiquitin chain on its a...

Low-barrier or short, strong hydrogen bonds have been proposed to contribute 10 to 20 kilocalories per mole to transition-state stabilization in enzymatic catalysis. The proposal invokes a large increase in hydrogen bond energy when the pKa values of the donor and acceptor (where Ka is the acid constant) become matched in the transition state (delta pKa=0). This hypothesis was tested by investigating the energetics of hydrogen bonds as a function of delta pKa for homologous series of compounds under nonaqueous conditions that are conducive to the formation of low-barrier hydrogen bonds. In all cases, there was a linear correlation between the increase in hydrogen-bond energy and the decrease in delta pKa, as expected from simple electrostatic effects. However, no additional energetic contribution to the hydrogen bond was observed at delta pKa=0. These results and those of other model studies suggest alternative mechanisms by which hydrogen bonds can contribute to enzymatic catalysis, in accord with conventional electrostatic considerations.

The ''GTPase switch'' paradigm, in which a GTPase switches between an active, GTP-bound state and an inactive, GDP-bound state through the recruitment of nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs), has been used to interpret the regulatory mechanism of many GTPases. A notable exception to this paradigm is provided by two GTPases in the signal recognition particle (SRP) and the SRP receptor (SR) that control the co-translational targeting of proteins to cellular membranes. Instead of the classical ''GTPase switch,'' both the SRP and SR undergo a series of discrete conformational rearrangements during their interaction with one another, culminating in their reciprocal GTPase activation. Here, we show that this series of rearrangements during SRP-SR binding and activation provide important control points to drive and regulate protein targeting. Using real-time fluorescence, we showed that the cargo for SRPribosomes translating nascent polypeptides with signal sequences-accelerates SRP⅐SR complex assembly over 100-fold, thereby driving rapid delivery of cargo to the membrane. A series of subsequent rearrangements in the SRP⅐SR GTPase complex provide important driving forces to unload the cargo during late stages of protein targeting. Further, the cargo delays GTPase activation in the SRP⅐SR complex by 8 -12 fold, creating an important time window that could further improve the efficiency and fidelity of protein targeting. Thus, the SRP and SR GTPases, without recruiting external regulatory factors, constitute a self-sufficient system that provides exquisite spatial and temporal control of a complex cellular process.conformational change ͉ fluorescence spectroscopy ͉ protein targeting and translocation ͉ signal recognition particle

Metal ions are critical for catalysis by many RNA and protein enzymes. To understand how these enzymes use metal ions for catalysis, it is crucial to determine how many metal ions are positioned at the active site. We report here an approach, combining atomic mutagenesis with quantitative determination of metal ion affinities, that allows individual metal ions to be distinguished. Using this approach, we show that at the active site of the Tetrahymena group I ribozyme the previously identified metal ion interactions with three substrate atoms, the 3-oxygen of the oligonucleotide substrate and the 3-and 2-moieties of the guanosine nucleophile, are mediated by three distinct metal ions. This approach provides a general tool for distinguishing active site metal ions and allows the properties and roles of individual metal ions to be probed, even within the sea of metal ions bound to RNA. D ivalent metal ions play a critical role in catalysis by many RNA and protein enzymes (e.g., see refs. 1-10). Determining the number of metal ions in an enzymatic active site and delineating their catalytic roles are crucial for elucidating the catalytic mechanisms of these enzymes (e.g., refs. 1-3, 5, 9, and 11-34). This presents a formidable challenge, especially for RNA enzymes, as the metal ions that directly participate in the chemical transformation are bound within a sea of metal ions that coat the charged RNA backbone and facilitate RNA folding ( Fig. 1; e.g., see refs. 32-39).The Tetrahymena group I ribozyme (E) derived from a selfsplicing group I intron catalyzes a reaction that mimics the first step of splicing, in which an exogenous guanosine nucleophile (G) cleaves a specific phosphodiester bond of an oligonucleotide substrate (S; Eq. 1; refs. 40-42).Three metal ion interactions contribute to catalysis by this ribozyme (Fig. 2). These interactions were previously identified by modification of specific substrate atoms that alter metal ion specificity; reactions with sulfur-or nitrogen-substituted substrates were severely compromised in Mg 2ϩ but were stimulated by addition of softer metal ions such as Mn 2ϩ (3,5,7,9). A fundamental question that remains unanswered, however, is whether these interactions are mediated by the same or by distinct metal ions.We report here the development of an approach that combines atomic-level substrate modifications with quantitative analyses to determine the affinity of individual metal ions for an enzymatic active site. These affinities provide a fingerprint for each metal ion, allowing distinct metal ions to be distinguished. Using this approach, we have provided evidence for three distinct metal ions within the active site of the Tetrahymena ribozyme. The results and the approach described herein will allow us to further probe the functional consequences of specific metal ion interactions and the catalytic role of individual metal ions, even within the sea of metal ions bound to RNA. Materials and MethodsMaterials. Ribozyme was prepared by in vitro transcription with T7 RNA polymera...