It takes two transducins to activate the cGMP-phosphodiesterase 6 in retinal rods.

Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme in G protein-coupled signal transduction. In retinal rods and cones, PDE6 is membrane-bound and activated to hydrolyse its substrate, cGMP, by binding of two active G protein α-subunits (Gα*). To investigate the activation mechanism of mammalian rod PDE6, we have collected functional and structural data, and analysed them by reaction-diffusion simulations. Gα* titration of membrane-bound PDE6 reveals a strong functional asymmetry of the enzyme with respect to the affinity of Gα* for its two binding sites on membrane-bound PDE6 and the enzymatic activity of the intermediary 1 : 1 Gα* · PDE6 complex. Employing cGMP and its 8-bromo analogue as substrates, we find that Gα* · PDE6 forms with high affinity but has virtually no cGMP hydrolytic activity. To fully activate PDE6, it takes a second copy of Gα* which binds with lower affinity, forming Gα* · PDE6 · Gα*. Reaction-diffusion simulations show that the functional asymmetry of membrane-bound PDE6 constitutes a coincidence switch and explains the lack of G protein-related noise in visual signal transduction. The high local concentration of Gα* generated by a light-activated rhodopsin molecule efficiently activates PDE6, whereas the low density of spontaneously activated Gα* fails to activate the effector enzyme.

Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement.

The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.

Structure and dynamics of the mammalian ribosomal pretranslocation complex.

Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.

Multiparticle cryo-EM of ribosomes.

As the resolution of cryo-EM reconstructions has improved to the subnanometer range, conformational and compositional heterogeneity have become increasing problems in cryo-EM, limiting the resolution of reconstructions. Since further purification is not feasible, the presence of several conformational states of ribosomal complexes in thermodynamic equilibrium requires methods for separating these states in silico. We describe a procedure for generating subnanometer resolution cryo-EM structures from large sets of projection images of ribosomal complexes. The incremental K-means-like method of unsupervised 3D sorting discussed here allows separation of classes in the dataset by exploiting intrinsic divisions in the data. The classification procedure is described in detail and its effectiveness is illustrated using current examples from our work. Through a good separation of conformational modes, higher resolution reconstructions can be calculated. This increases information gained from single states, while exploiting the coexistence of multiple states to gather comprehensive mechanistic insight into biological processes like ribosomal translocation.

Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome.

RATIONALE: Repair mechanisms resulting in alveolar protein degradation in acute respiratory distress syndrome (ARDS) are largely unknown. OBJECTIVES: To test whether the 20S proteasome is present and functional in the alveolar space in patients with ARDS. METHODS: Proteasome antigenic concentration in bronchoalveolar lavage (BAL) supernatants was measured by ELISA in patients with ARDS (n = 64), acute lung injury (ALI) (n = 8), sarcoidosis (n = 13), and in healthy subjects (n = 8). Cleavage of specific fluorogenic substrates (+/-epoxomicin), I(125) albumin degradation rate, and gel filtration were used to quantify and characterize proteasomal activity. The presence of proteasomes was confirmed independently by electron microscopic techniques. MEASUREMENTS AND MAIN RESULTS: Proteasome concentrations in patients with ARDS were markedly increased (1,069 +/- 1,194 ng/ml) in comparison to healthy subjects (60.8 +/- 49.8; P < 0.001), ALI (154 +/- 43; P = 0.006), and sarcoidosis (97.6 +/- 42.2; P = 0.037). All fluorogenic substrates were hydrolyzed (Suc-LLVY-AMC, 3.6 +/- 8.8 pkat/mg; BZ-VGR-AMC, 1.8 +/- 3.1; Suc-LLE-AMC, 1 +/- 1.7) by BAL supernatants of patients with ARDS, with inhibition by epoxomicin (P = 0.0001), and the majority of proteolytic activity was detected in BAL supernatant. Maximum hydrolyzing activity occurred at 660 kD and 20S proteasome was seen microscopically after purification and being released by pneumocytes type II. Proteasomal activity and albumin degradation rate in patients with ARDS were approximately 17-fold lower than in healthy subjects. Proteasomal activity in normal BAL was inhibited by BAL aliquots from patients with ARDS but not by denatured BAL, and returned to normal by purification. CONCLUSIONS: For the first time, we identified extracellular, biologically active 20S proteasome in the alveolar space of patients with ARDS in concentrations much higher than in normal subjects or in those with ALI.

GTPase activation of elongation factor EF-Tu by the ribosome during decoding.

We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors.

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.

Structure of the ribosome-bound cricket paralysis virus IRES RNA.

Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.