Bacillus subtilis RNA deprotection enzyme RppH recognizes guanosine in the second position of its substrates.

The initiation of mRNA degradation often requires deprotection of its 5' end. In eukaryotes, the 5'-methylguanosine (cap) structure is principally removed by the Nudix family decapping enzyme Dcp2, yielding a 5'-monophosphorylated RNA that is a substrate for 5' exoribonucleases. In bacteria, the 5'-triphosphate group of primary transcripts is also converted to a 5' monophosphate by a Nudix protein called RNA pyrophosphohydrolase (RppH), allowing access to both endo- and 5' exoribonucleases. Here we present the crystal structures of Bacillus subtilis RppH (BsRppH) bound to GTP and to a triphosphorylated dinucleotide RNA. In contrast to Bdellovibrio bacteriovorus RppH, which recognizes the first nucleotide of its RNA targets, the B. subtilis enzyme has a binding pocket that prefers guanosine residues in the second position of its substrates. The identification of sequence specificity for RppH in an internal position was a highly unexpected result. NMR chemical shift mapping in solution shows that at least three nucleotides are required for unambiguous binding of RNA. Biochemical assays of BsRppH on RNA substrates with single-base-mutation changes in the first four nucleotides confirm the importance of guanosine in position two for optimal enzyme activity. Our experiments highlight important structural and functional differences between BsRppH and the RNA deprotection enzymes of distantly related bacteria.

Design and characterization of modular scaffolds for tubulin assembly.

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin.

Structural changes occur in the alphabeta-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a beta-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with alpha-tubulin from stacking onto a beta-tubulin beta sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the beta-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.

Stathmin and interfacial microtubule inhibitors recognize a naturally curved conformation of tubulin dimers.

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T(2)RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T(2)RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T(2)RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T(2)RB3, T(2)Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.

Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour.

Ribonucleases J1 and J2 are recently discovered enzymes with dual 5'-to-3' exoribonucleolytic/endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5'-to-3' exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

Studying drug-tubulin interactions by X-ray crystallography.

Tubulin, the microtubule building-block, is the target of numerous small molecule compounds that interfere with microtubule dynamics. Several of these ligands are in clinical use as antitumor drugs. There have been numerous studies on these molecules, with two main objectives: to determine their mechanism of action and to find new compounds that would expand the arsenal available for cancer chemotherapy. Although these studies would undoubtedly benefit from structural data on tubulin, this protein has long resisted crystallization attempts. We have used stathmin-like domains (SLDs) of stathmin family proteins as a tool to crystallize tubulin and have obtained three-dimensional crystals of the tubulin:SLD complexes. As many tubulin ligands bind to these complexes, the crystals are valuable tools to study tubulin-drug interactions by X-ray crystallography. They open the way to a structure-based drug design approach.

Molecular basis for the recognition and cleavage of RNA by the bifunctional 5'-3' exo/endoribonuclease RNase J.

RNase J is a key member of the ß-CASP family of metallo-ß-lactamases involved in the maturation and turnover of RNAs in prokaryotes. The B. subtilis enzyme possesses both 5'-3' exoribonucleolytic and endonucleolytic activity, an unusual property for a ribonuclease. Here, we present the crystal structure of T. thermophilus RNase J bound to a 4 nucleotide RNA. The structure reveals an RNA-binding channel that illustrates how the enzyme functions in 5'-3' exoribonucleolytic mode and how it can function as an endonuclease. A second, negatively charged tunnel leads from the active site, and is ideally located to evacuate the cleaved nucleotide in 5'-3' exonucleolytic mode. We show that B. subtilis RNase J1, which shows processive behavior on long RNAs, behaves distributively for substrates less than 5 nucleotides in length. We propose a model involving the binding of the RNA to the surface of the ß-CASP domain to explain the enzyme's processive action.