Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.cytoplasmic dynein | mechanosensing | optical tweezers | AAA+ ATPases | microtubules N umerous eukaryotic cellular processes require motion and force generated by cytoskeletal motor proteins, among which cytoplasmic dynein (hereinafter, "dynein") is unique for its size, complexity, and versatility. As a homodimeric, divergent AAA+ ATPase (AAA: ATPase associated with various cellular activities), dynein drives the majority of microtubule (MT) minusend-directed motility in most eukaryotes (1). The motor functions as a massive protein complex (2), but its catalytic core consists of two identical heavy chains, each with six AAA modules (AAA1-6) linked in tandem to form a ring (Fig. 1A). AAA1-4 bind nucleotides, whereas AAA5 and -6 are structural (3, 4). A ∼15-nm "stalk" emerging from AAA4 (3, 4) separates the AAA modules from the MT-binding domain (MTBD). The stalk configuration influences both MT affinity and ATPase activity (5) and thereby mediates bidirectional allosteric communication between the AAA ring and the MTBD (3, 6). Finally, a ∼10-nm "linker" also emerges from the ring and undergoes cyclic reorientations that generate force and displacement (7-9).For dynein to "walk," one motor domain ("head") must remain MT-bound while the other moves (10-13), thus requiring coordination of the "internal" cycles of both heads. Dynein may use allosteric mechanosensing (possibly through the stalk) to differentiate be...

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s ؊1 , regardless of the tRNA identity. The reactivity decreased in the order Lys ‫؍‬ Arg > Ala > Ser > Phe ‫؍‬ Val > Asp Ͼ Ͼ Pro, with Pro being by far the slowest. However, when Phe-tRNA Phe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ϳ7 s ؊1 , which corresponds to the rate of binding of Phe-tRNA Phe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyltRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.The enzymatic activity of the ribosome is to catalyze peptide bond formation. During the peptidyl transfer reaction, the ␣-amino group of aminoacyl-tRNA bound to the A site of the ribosome attacks the ester bond of peptidyl-tRNA in the P site, which results in peptidyl-tRNA extended by one amino acid in the A site and deacylated tRNA in the P site. The tRNA substrates are aligned in the active center of the ribosome by interactions of their CCA ends with 23 S rRNA bases (1-3). The ribosome lowers the activation entropy of the reaction (4, 5) by orienting the reacting groups precisely relative to each other (2, 3), providing an electrostatic environment that reduces the free energy of forming the transition state, shielding the reaction against bulk water (6, 7), or a combination of these effects (8).The peptidyl transfer reaction is modulated by conformational changes at the active site (3, 8 -10) as well as by the nature of the substrates. Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19). Early studies with 50 S ribosomal subunits indicated that efficient peptidyl transfer was observed with 3Ј-terminal RNase T1 fragments of N-acetylArg-tRNA Arg and fMet-tRNA fMet as model P-site substrates and an analog of aminoacyl-tRNA, puromycin (Pmn 4 ; O-methyltyrosine linked to N 6 -dimethyladenosine via an amide bond), as A-site substrate (20). In contrast,...

The reduction of nitrite (NO 2 ؊ ) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ؎ 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H 2 O and D 2 O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ϳ1.33 ؎ 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substratefree AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H 2 O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mM. As a consequence, the observed ET process was faster in D 2 O than in H 2 O above 0.1 mM nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ϳ0.61 ؎ 0.02 between 3 and 10 mM. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.

Conformational control limits most electron transfer (ET) reactions in biology, but we lack general insight into the extent of conformational space explored, and specifically the properties of the associated energy landscape. Here we unite electron-electron double resonance (ELDOR) studies of the diradical (disemiquinoid) form of human cytochrome P450 reductase (CPR), a nicotinamide adenine phosphate dinucleotide (NADPH)-linked diflavin oxidoreductase required for P450 enzyme reduction, with functional studies of internal ET to gain new insight into the extent and properties of the energy landscape for conformationally controlled ET. We have identified multiple conformations of disemiquinoid CPR, which point to a rugged energy landscape for conformational sampling consistent with functional analysis of ET using high-pressure stopped-flow, solvent, and temperature perturbation studies. Crystal structures of CPR have identified discrete "closed" and "open" states, but we emphasize the importance of a continuum of conformational states across the energy landscape. Within the landscape more closed states that favor internal ET are formed by nucleotide binding. Open states that enable P450 enzymes to gain access to electrons located in the FMN-domain are favored in the absence of bound coenzyme. The extent and nature of energy landscapes are therefore accessible through the integration of ELDOR spectroscopy with functional studies. We suggest this is a general approach that can be used to gain new insight into energy landscapes for biological ET mediated by conformational sampling mechanisms.

Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.