Browsing by Subject "Hydrolysis"
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Item Open Access A Comprehensive Study of Guanosine-5'-triphosphate Hydrolysis by the Bacterial Cell Division Protein FtsZ(2018) Salsburg, AndrewThe bacterial protein FtsZ plays a vital role in cytokinesis in prokaryotes as it polymerizes to form an FtsZ ring (Z ring) at the division septum midcell. FtsZ exhibits a GTP hydrolysis activity and attempts have been made to model the kinetics of this process. There is a major discrepancy, however, over the concentration of GTP needed for activity. The dissociation constant (KD) between wild-type FtsZ protein and GTP was measured to be 30 nM using isothermal titration calorimetry. In contrast, several research groups have reported that GTP hydrolysis required GTP concentrations in the millimolar range. They used Michaelis-Menten kinetics to model the GTP concentration dependence and obtained an apparent binding constant (Km) in the range of 300-1,000 µM GTP. Km and KD are not identical measures of binding given that they differ by the kinetic constant governing catalysis, kcat, but we suggest that a five order of magnitude difference between the values is unprecedented and this was a problem that needed investigating.
My overall goal in this work has been to perform a comprehensive in vitro study of the FtsZ GTP hydrolysis activity using an enzyme-coupled regenerating system. With this system rates of GTP hydrolysis by FtsZ are obtained spectrophotometrically. I have confirmed for wild-type FtsZ that GTP hydrolysis rates show little to no dependence on GTP concentrations in the range of 50-3,000 µM, contradicting the high values of Km reported in some previous studies. Since we have failed to reproduce the high Km with three different preparations of FtsZ protein, we cannot propose a definitive mechanism for the previous results.
I also measured the GTP hydrolysis of several mutants of FtsZ: E238A, L169R, and FtsZ84 (G105S). I investigated each of these mutants to see if they had a high apparent Km. FtsZ84 had a low overall hydrolysis rate, but did show a large increase in hydrolysis rates when GTP was increased from 50-3,000 µM. We hypothesize that a lower affinity for GTP is not a Michaelis-Menten Km, but likely a reflection of a weak binding of GTP by FtsZ84, giving KD in the millimolar range.
Item Open Access A kinesin motor in a force-producing conformation.(BMC Struct Biol, 2010-07-05) Heuston, Elisabeth; Bronner, C Eric; Kull, F Jon; Endow, Sharyn ABACKGROUND: Kinesin motors hydrolyze ATP to produce force and move along microtubules, converting chemical energy into work by a mechanism that is only poorly understood. Key transitions and intermediate states in the process are still structurally uncharacterized, and remain outstanding questions in the field. Perturbing the motor by introducing point mutations could stabilize transitional or unstable states, providing critical information about these rarer states. RESULTS: Here we show that mutation of a single residue in the kinesin-14 Ncd causes the motor to release ADP and hydrolyze ATP faster than wild type, but move more slowly along microtubules in gliding assays, uncoupling nucleotide hydrolysis from force generation. A crystal structure of the motor shows a large rotation of the stalk, a conformation representing a force-producing stroke of Ncd. Three C-terminal residues of Ncd, visible for the first time, interact with the central beta-sheet and dock onto the motor core, forming a structure resembling the kinesin-1 neck linker, which has been proposed to be the primary force-generating mechanical element of kinesin-1. CONCLUSIONS: Force generation by minus-end Ncd involves docking of the C-terminus, which forms a structure resembling the kinesin-1 neck linker. The mechanism by which the plus- and minus-end motors produce force to move to opposite ends of the microtubule appears to involve the same conformational changes, but distinct structural linkers. Unstable ADP binding may destabilize the motor-ADP state, triggering Ncd stalk rotation and C-terminus docking, producing a working stroke of the motor.Item Open Access Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor.(Nat Commun, 2014-06-17) Migliori, Amy D; Keller, Nicholas; Alam, Tanfis I; Mahalingam, Marthandan; Rao, Venigalla B; Arya, Gaurav; Smith, Douglas EHow viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips. We further show that the measured impairments correlate with computed changes in free-energy differences between the two states. These findings support the proposed structural mechanism and further suggest an energy landscape model of motor activity that couples the free-energy profile of motor conformational states with that of the ATP hydrolysis cycle.Item Open Access Inside-out Z rings--constriction with and without GTP hydrolysis.(Molecular microbiology, 2011-07) Osawa, Masaki; Erickson, Harold PThe bacterial tubulin homologue FtsZ forms a ring-like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ-YFP-mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts-FtsZ-YFP, where the membrane tether is switched to the opposite side of the protofilament. This assembled 'inside-out' Z rings that wrapped around the outside surface of tubular liposomes. The inside-out Z rings were highly dynamic, and generated a constriction force that squeezed the tubular liposomes from outside. This is consistent with models where the constriction force is generated by curved protofilaments bending the membrane. We used this system to test how GTP hydrolysis by FtsZ is involved in Z-ring constriction. Without GTP hydrolysis, Z rings could still assemble and generate an initial constriction. However, the constriction quickly stopped, suggesting that Z rings became rigidly stabilized in the absence of GTP hydrolysis. We propose that remodelling of the Z ring, mediated by GTP hydrolysis and exchange of subunits, is necessary for the continuous constriction.Item Open Access Rapid in vitro assembly of Caulobacter crescentus FtsZ protein at pH 6.5 and 7.2.(The Journal of biological chemistry, 2013-08) Milam, Sara L; Erickson, Harold PFtsZ from most bacteria assembles rapidly in vitro, reaching a steady-state plateau in 5-10 s after addition of GTP. A recent study used a novel dynamic light-scattering technique to assay the assembly of FtsZ from Caulobacter crescentus (CcFtsZ) and reported that assembly required 10 min, ∼100 times slower than for related bacteria. Previous studies had indicated normal, rapid assembly of CcFtsZ. We have reinvestigated the assembly kinetics using a mutant L72W, where assembly of subunits into protofilaments results in a significant increase in tryptophan fluorescence. We found that assembly reached a plateau in 5-10 s and showed no change in the following 10 min. This was confirmed by 90° light scattering and negative-stain electron microscopy. The very slow kinetics in the dynamic light-scattering study may be related to a refractory state induced when the FtsZ protein is stored without nucleotide, a phenomenon that we had observed in a previous study of EcFtsZ. We conclude that CcFtsZ is not an outlier, but shows rapid assembly kinetics similar to FtsZ from related bacteria.Item Open Access Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function.(Proc Natl Acad Sci U S A, 1990-04) Cotecchia, S; Exum, S; Caron, MG; Lefkowitz, RJRegions of the hamster alpha 1-adrenergic receptor (alpha 1 AR) that are important in GTP-binding protein (G protein)-mediated activation of phospholipase C were determined by studying the biological functions of mutant receptors constructed by recombinant DNA techniques. A chimeric receptor consisting of the beta 2-adrenergic receptor (beta 2AR) into which the putative third cytoplasmic loop of the alpha 1AR had been placed activated phosphatidylinositol metabolism as effectively as the native alpha 1AR, as did a truncated alpha 1AR lacking the last 47 residues in its cytoplasmic tail. Substitutions of beta 2AR amino acid sequence in the intermediate portions of the third cytoplasmic loop of the alpha 1AR or at the N-terminal portion of the cytoplasmic tail caused marked decreases in receptor coupling to phospholipase C. Conservative substitutions of two residues in the C terminus of the third cytoplasmic loop (Ala293----Leu, Lys290----His) increased the potency of agonists for stimulating phosphatidylinositol metabolism by up to 2 orders of magnitude. These data indicate (i) that the regions of the alpha 1AR that determine coupling to phosphatidylinositol metabolism are similar to those previously shown to be involved in coupling of beta 2AR to adenylate cyclase stimulation and (ii) that point mutations of a G-protein-coupled receptor can cause remarkable increases in sensitivity of biological response.Item Open Access Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.(PLoS One, 2008-01-16) Bennett, Craig B; Westmoreland, Tammy J; Verrier, Carmel S; Blanchette, Carrie AB; Sabin, Tiffany L; Phatnani, Hemali P; Mishina, Yuliya V; Huper, Gudrun; Selim, Alice L; Madison, Ernest R; Bailey, Dominique D; Falae, Adebola I; Galli, Alvaro; Olson, John A; Greenleaf, Arno L; Marks, Jeffrey RBRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.