Chemical-level details such as protonation and hybridization state are critical for understanding enzyme mechanism and function. Even at high resolution, these details are difficult to determine by X-ray crystallography alone. The chemical shift in NMR spectroscopy, however, is an extremely sensitive probe of the chemical environment, making solid-state NMR spectroscopy and X-ray crystallography a powerful combination for defining chemically detailed three-dimensional structures. Here we adopted this combined approach to determine the chemically rich crystal structure of the indoline quinonoid intermediate in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. Models of the active site were developed using a synergistic approach in which the structure of this reactive substrate analogue was optimized using ab initio computational chemistry in the presence of side-chain residues fixed at their crystallographically determined coordinates. Various models of charge and protonation state for the substrate and nearby catalytic residues could be uniquely distinguished by their calculated effects on the chemical shifts measured at specifically (13)C- and (15)N-labeled positions on the substrate. Our model suggests the importance of an equilibrium between tautomeric forms of the substrate, with the protonation state of the major isomer directing the next catalytic step.

Helicases are ubiquitous ATPases with widespread roles in genome metabolism. Here we report a new functionality for ATPases with helicase-like domains, namely that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. This study defines the mechanism for these enzymes and shows how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism, for example, in nucleotide excision and mismatch repair.Helicases were defined classically as ATPases that use directional translocation to unwind duplex nucleic acids. More recently however, many "pseudo-helicases" have been described that posses amino acid motifs characteristic of helicases yet fulfill their cellular functions without obligate DNA unwinding (1) and instead are often translocases that move directionally on single-(2) or double-stranded (3,4) nucleic acids. Pseudo-helicases include the ATP-dependent bacterial Type I and III restriction enzymes (REs) which use ATP hydrolysis to communicate between two distant restriction sites on the same DNA (5,6). Only if both sites are unmethylated is the DNA considered "foreign" and destroyed by the endonuclease. Type III REs form a heterooligomeric complex of two subunits: Mod (for target site methylation) and Res (for ATPase and endonuclease activities). Because they only hemimethylate their asymmetric recognition sequences, these enzymes must signal the relative site orientation during communication (7): cleavage occurs only if the sites are in an inverted-repeat orientation, arranged either "head-to-head" (HtH) (7) or "tail-to-tail" (TtT) (8). Type III REs hydrolyze only a few tens of ATP per dsDNA break (9,10), suggesting either passive three-dimensional looping between sites with limited translocation (11) or diffusion in 1D following an ATP-dependent switch (Fig. S1A) To distinguish between the different communication models, we developed a real-time single-molecule assay using magnetic tweezers combined with TIRF microscopy (12) that can localize fluorescently-labeled enzymes on stretched 26 kbp DNA molecules containing two centrally-located HtH EcoP15I sites with 6 kbp intersite distance (Fig. 1A). EcoP15I was labeled with quantum dots on the C-terminus of the Res subunit (QR-EcoP15I), without compromising the biochemical activity (Fig. S2). While DNA binding by lone quantum dots was not detected, addition of QR-EcoP15I and ATP resulted in enzyme attachment at one ( Fig. 1B and movie S1) or two ( Fig. S3) specific recognition sites. Enzymes always arrived instantaneously at the sites, with no evidence for long-lived non-specific binding en route (N = 61). Following a delay of, on av...

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted "membrane-anchored" gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surfaceimmobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport. molecular motors | lipid bilayers | transport efficiency | motor-cargo coupling | streptavidin-binding peptide I ntracellular transport of membrane-bound vesicles and organelles is a process fundamental to many cellular functions including morphogenesis, signaling, and growth (1-4). Active cargo transport inside eukaryotic cells is mediated by ensembles of motor proteins, such as kinesins and dynein, walking on microtubule tracks (5), and myosins walking on actin filaments (6). Gaining mechanistic insight into the functioning of these motors inside the complex environment of cells is challenging. Several studies have thus used in vitro approaches to investigate transport mediated by groups of same or different motors attached to cargos such as silica beads (7), quantum dots (8), glass coverslips (9, 10), or DNA scaffolds (11,12). Although these approaches provide us with knowledge about the collective dynamics of multimotor transport, a key anomaly in these in vitro systems is the use of rather nonphysiological rigid cargo. Vesicular cargo transport by molecular motors requires their attachment to a fluid lipid bilayer either directly or via different adaptor molecules. The anchoring of motors in a diffusive lipid environment induces loose intermotor coupling along with the motors diffusing within the lipid bilayer, thereby increasing the flexibility of the system....

Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at constant velocity until no overlap between the microtubules remains, leading to breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14, HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback by the local interaction kinetics of HSET with overlapping microtubules, causing retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force antagonizing the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through local alteration of their individual interaction kinetics.