[show abstract][hide abstract] ABSTRACT: The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.
[show abstract][hide abstract] ABSTRACT: Kinesin-5, a widely conserved motor protein required for assembly of the bipolar mitotic spindle in eukaryotes, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell-shaped molecule [1-3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5's ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a nonmotor microtubule binding site in its C terminus . However, it is not known how the nonmotor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four nonmotor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the nonmotor C terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5's microtubule binding without substantially reducing motor activity. Our results suggest that tetramerization of kinesin-5's low-processivity motor domains is not sufficient for microtubule sliding because the motor domains alone are unlikely to maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes nonmotor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.
Current biology: CB 01/2011; 21(2):154-60. · 10.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.
The Journal of Cell Biology 09/2008; 182(3):421-8. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The free 2'-3' cis-diol at the 3'-terminus of tRNA provides a unique juxtaposition of functional groups that play critical roles during protein synthesis. The translation process involves universally conserved chemistry at almost every stage of this multistep procedure, and the 2'- and 3'-OHs are in the immediate vicinity of chemistry at each step. The cis-diol contribution affects steps ranging from tRNA aminoacylation to peptide bond formation. The contributions have been studied in assays related to translation over a period that spans at least three decades. In this review, we follow the 2'- and 3'-OHs through the steps of translation and examine the involvement of these critical functional groups.
[show abstract][hide abstract] ABSTRACT: The ribosomal peptidyl transferase center is expected to be regiospecific with regard to its tRNA substrates, yet the ester linkages between the tRNA and the amino acid or peptide are susceptible to isomerization between the O2' and O3' hydroxyls of the terminal A76 ribose sugar. To establish which isomer of the P site tRNA ester is utilized by the ribosome, we prepared two nonisomerizable transition state inhibitors with either an A76 O2' or O3' linkage. Strong preferential binding to the O3' regioisomer indicates that the peptidyl transferase proceeds through a transition state with an O3'-linked peptide in the P-site.
Journal of the American Chemical Society 04/2006; 128(10):3108-9. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.
[show abstract][hide abstract] ABSTRACT: All living cells are dependent on ribosomes to catalyze the peptidyl transfer reaction, by which amino acids are assembled into proteins. The previously studied peptidyl transferase transition state analog CC-dA-phosphate-puromycin (CCdApPmn) has important differences from the transition state, yet current models of the ribosomal active site have been heavily influenced by the properties of this molecule. One significant difference is the substitution of deoxyadenosine for riboadenosine at A76, which mimics the 3' end of a P-site tRNA. We have developed a solid phase synthetic approach to produce inhibitors that more closely match the transition state, including the critical P-site 2'-OH. Inclusion of the 2'-OH or an even bulkier OCH3 group causes significant changes in binding affinity. We also investigated the effects of changing the A-site amino acid side chain from phenylalanine to alanine. These results indicate that the absence of the 2'-OH is likely to play a significant role in the binding and conformation of CCdApPmn in the ribosomal active site by eliminating steric clash between the 2'-OH and the tetrahedral phosphate oxygen. The conformation of the actual transition state must allow for the presence of the 2'-OH, and transition state mimics that include this critical hydroxyl group must bind in a different conformation from that seen in prior analog structures. These new inhibitors will provide valuable insights into the geometry and mechanism of the ribosomal active site.
Nucleic Acids Research 02/2004; 32(4):1502-11. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Known sequence motifs containing key glycine residues can drive the homo-oligomerization of transmembrane helices. To find other motifs, a randomized library of transmembrane interfaces was generated in which glycine was omitted. The TOXCAT system, which measures transmembrane helix association in the Escherichia coli inner membrane, was used to select high-affinity homo-oligomerizing sequences in this library. The two most frequently occurring motifs were SxxSSxxT and SxxxSSxxT. Isosteric mutations of any one of the serine and threonine residues to non-polar residues abolished oligomerization, indicating that the interaction between these positions is specific and requires an extended motif of serine and threonine hydroxyl groups. Computational modeling of these sequences produced several chemically plausible structures that contain multiple hydrogen bonds between the serine and threonine residues. While single serine or threonine side-chains do not appear to promote helix association, motifs can drive strong and specific association through a cooperative network of interhelical hydrogen bonds.
Journal of Molecular Biology 03/2002; 316(3):799-805. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Motor proteins move cargos in cells and are involved in coordinated large-scale force generation e.g. in cell locomotion,
cell division or muscle contraction. Optical traps in conjunction with high-resolution light microscopy has been a tool of
choice to analyze dynamic properties of motors, such as power strokes, step lengths or unitary forces. Much detail about microscopic
function has also been learned by direct imaging.
[show abstract][hide abstract] ABSTRACT: Translation, the synthesis of proteins from individual amino acids based on genetic information, is a cornerstone biological process. During ribosomal protein synthesis, new peptide bonds form through aminolysis of the peptidyl-tRNA ester bond by the alpha-amino group of the A-site amino acid. The rate of this reaction is accelerated at least 10(7)-fold in the ribosome, but the catalytic mechanism has remained controversial. We have used a combination of synthetic chemistry, biochemical, and structural biology approaches to characterize the mechanism of the peptidyl transfer reaction and the configuration of the reaction's tetrahedral intermediate. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. This indicates that the 2'-OH is essential to the reaction through participation in substrate assisted catalysis. A series of novel mimics of the tetrahedral intermediate were examined to distinguish between possible regio- and stereoisomeric forms of the intermediate. The determination of these parameters has important implications for the configuration of the substrates and intermediate within the ribosomal active site, and thus which functional groups are properly positioned to play various roles in promoting the reaction. Our results contribute to an emerging model of the peptidyl transfer reaction in which the ribosomal active site positions the substrates in an orientation specifically designed to promote the reaction, wherein the A76 2'-OH serves as a proton shuttle to enable critical proton transfers in the formation of the final peptide product.
Blood Cells Molecules and Diseases 38(2):110-6. · 2.26 Impact Factor