Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.

Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.

Directed evolution of multivalent glycopeptides tightly recognized by HIV antibody 2G12.

Herein, we report a method for in vitro selection of multivalent glycopeptides, combining mRNA display with incorporation of unnatural amino acids and "click" chemistry. We have demonstrated the use of this method to design potential glycopeptide vaccines against HIV. From libraries of ~10(13) glycopeptides containing multiple Man9 glycan(s), we selected variants that bind to HIV broadly neutralizing antibody 2G12 with picomolar to low nanomolar affinity. This is comparable to the strength of the natural 2G12-gp120 interaction, and is the strongest affinity achieved to date with constructs containing 3-5 glycans. These glycopeptides are therefore of great interest in HIV vaccine design.

Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP dehydrogenase.

IMP dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the reduction of NAD(+). The reaction involves formation of an E-XMP covalent intermediate; hydrolysis of the E-XMP intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700-11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of K(R) ranging from 40 to 400 mM. The value of k(rescue) for the best rescue agents approaches the value of k(cat) for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that the guanidine derivatives do not restore the equilibrium between open and closed conformations. Therefore, rescue agents must accelerate the hydrolysis of the E-XMP intermediate. The rate of the rescue reaction increases with an increase in pH, consistent with the hypothesis that the reaction involves neutral guanidine. A solvent D(2)O isotope effect is observed at low concentrations of the rescue agent, consistent with rate-limiting transfer of a proton from water. The value of k(cat) (rescue)/K(R)(base) correlates with the pK(a) of the guanidine derivative (Bronsted coefficient beta approximately 1). These results suggest that proton transfer from water to guanidine is almost complete in the transition state.

Is Arg418 the catalytic base required for the hydrolysis step of the IMP dehydrogenase reaction?

The first committed step of guanine nucleotide biosynthesis is the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) catalyzed by IMP dehydrogenase. The reaction involves the reduction of NAD(+) with the formation of a covalent enzyme intermediate (E-XMP). Hydrolysis of E-XMP requires the enzyme to adopt a closed conformation and is rate-limiting. Thr321, Arg418, and Tyr419 are candidates for the residue that activates water. The substitution of Thr321 has similar, but small, effects on both the hydride transfer and hydrolysis steps. This result suggests that Thr321 influences the reactivity of Cys319, either through a direct interaction or by stabilizing the structure of the active site loop. The hydrolysis of E-XMP is accelerated by the deprotonation of a residue with a pK(a) of approximately 8. A similar deprotonation stabilizes the closed conformation; this residue has a pK(a) of >or=6 in the closed conformation. The substitution of Tyr419 with Phe does not change the pH dependence of either the hydrolysis of E-XMP or the conformational change, which suggests that Tyr419 is not the residue that activates water. In contrast, the conformational change becomes pH-independent when Arg418 is substituted with Gln. Lys can replace the function of Arg418 in the hydrolysis reaction but does not stabilize the closed conformation. The simplest explanation for these observations is that Arg418 serves as the base that activates water in the IMPDH reaction.

A twisted base? The role of arginine in enzyme-catalyzed proton abstractions.

Arginine residues are generally considered poor candidates for the role of general bases because they are predominantly protonated at physiological pH. Nonetheless, Arg residues have recently emerged as general bases in several enzymes: IMP dehydrogenase, pectate/pectin lyases, fumarate reductase, and l-aspartate oxidase. The experimental evidence suggesting this mechanistic function is reviewed. Although these enzymes have several different folds and distinct evolutionary origins, a common structural motif is found where the critical Arg residue is solvent accessible and adjacent to carboxylate groups. The chemistry of the guanidine group suggests unique strategies to lower the pK(a) of Arg. Lastly, the presumption that general bases must be predominantly deprotonated is revisited.

Substitution of the conserved Arg-Tyr dyad selectively disrupts the hydrolysis phase of the IMP dehydrogenase reaction.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP via the covalent E-XMP* intermediate (E-XMP*), with the concomitant reduction of NAD(+). Hydrolysis of E-XMP* is rate-limiting, and the catalytic base required for this step has not been identified. An X-ray crystal structure of Tritrichomonas foetus IMPDH with mizoribine monophosphate (MZP) reveals a novel closed conformation in which a mobile flap occupies the NAD(+)/NADH site [Gan, L., Seyedsayamdost, M. R., Shuto, S., Matsuda, A., Petsko, G. A., and Hedstrom, L. (2003) Biochemistry 42, 857-863]. In this complex, a water molecule is coordinated between flap residues Arg418 and Tyr419 and MZP in a geometry that resembles the transition state for hydrolysis of E-XMP*, which suggests that the Arg418-Tyr419 dyad activates water. We constructed and characterized two point mutants, Arg418Ala and Tyr419Phe, to probe the role of the Arg418-Tyr419 dyad in the IMPDH reaction. Arg418Ala and Tyr419Phe decrease k(cat) by factors of 500 and 10, respectively, but have no effect on hydride transfer or NADH release. In addition, the mutants display increased solvent isotope effects and increased levels of steady-state accumulation of E-XMP*. Inhibitor analysis indicates that the mutations destabilize the closed conformation, but this effect can account for a decrease in k(cat) of no more than a factor of 2. These observations demonstrate that both the Arg418Ala and Tyr419Phe mutations selectively impair hydrolysis of E-XMP* by disrupting the chemical transformation. Moreover, since the effects of the Tyr419Phe mutation are comparatively small, these experiments suggest that Arg418 acts as the base to activate water.