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November 24, 2007

Nikolett Nagy wins 2nd prize at the Student Research Conference of Eötvös University

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September 21-22, 2007

Joint retreat conference with Dr. András Málnási-Csizmadia’s research group, held in Balatonföldvár, Hungary

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September 13, 2007

Paper published in J. Biol. Chem. about the mechanism of action of the human dUTPase enzyme

Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme.

Tóth J, Varga B, Kovács M, Málnási-Csizmadia A, Vértessy BG.

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined 3D structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: i) fast substrate binding, ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, iii) hydrolysis (chemical) step iv) and rapid, non-ordered release of the products. Independent quench-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quench-flow, we devised a novel method to synthesize gamma32P-labelled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

PubMed Abstract




June 4, 2007

Paper published in PNAS about load-dependent mechanism of non-muscle myosins 2A and 2B

Load-dependent mechanism of nonmuscle myosin 2.

Kovács M, Thirumurugan K, Knight PJ, Sellers JR.

Loads on molecular motors regulate and coordinate their function. In a study that directly measures properties of internally strained myosin 2 heads bound to actin, we find that human nonmuscle myosins 2A and 2B show marked load-dependent changes in kinetics of ADP release but not in nucleotide binding. We show that the ADP release rate constant is increased 4-fold by the assisting load on one head and decreased 5-fold (for 2A) or 12-fold (for 2B) by the resisting load on the other. Thus these myosins, especially 2B, have marked mechanosensitivity of product release. By regulating the actin attachment of myosin heads, this provides a basis for energy-efficient tension maintenance without obstructing cellular contractility driven by other motors such as smooth muscle myosin. Whereas forward load accelerates the cycle of interaction with actin, resistive load increases duty ratio to favor tension maintenance by two-headed attachment.

PubMed Abstract




May 15, 2007

Paper published in Structure about strong actin-binding structures of molluscan myosins, indicating correspondence between cleft closure and thermodynamics of actin binding

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

Yang Y, Gourinath S, Kovács M, Nyitray L, Reutzel R, Himmel DM, O'Neall-Hennessey E, Reshetnikova L, Szent-Györgyi AG, Brown JH, Cohen C.

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

PubMed Abstract
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1 May, 2007

New website launched



April 21, 2007

Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release.

Malnasi-Csizmadia A, Toth J, Pearson DS, Hetenyi C, Nyitray L, Geeves MA, Bagshaw CR, Kovacs M.

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the powerstroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys84 and Arg704, we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.

PubMed Abstract



March 20, 2007

Diversity of structural behavior in vertebrate conventional myosins complexed with actin.

Iwamoto H, Oiwa K, Kovacs M, Sellers JR, Suzuki T, Wakayama J, Tamura T, Yagi N, Fujisawa T.

Low-resolution three-dimensional structures of acto-myosin subfragment-1 (S1) complexes were retrieved from X-ray fiber diffraction patterns, recorded either in the presence or absence of ADP. The S1 was obtained from various myosin-II isoforms from vertebrates, including rabbit fast-skeletal and cardiac, chicken smooth and human non-muscle IIA and IIB species, and was diffused into an array of overstretched, skinned skeletal muscle fibers. The S1 attached to the exposed actin filaments according to their helical symmetry. Upon addition of ADP, the diffraction patterns from acto-S1 showed an increasing magnitude of response in the order as listed above, with features of a lateral compression of the whole diffraction pattern (indicative of increased radius of the acto-S1 complex) and an enhancement of the fifth layer-line reflection. The structure retrieval indicates that these changes are mainly due to the swing of the light chain (LC) domain in the direction consistent with the cryo-electron microscopic results. In the non-muscle isoforms, the swing is large enough to affect the manner of quasi-crystal packing of the S1-decorated actin filaments and their lattice dimension, with a small change in the twist of actin filaments. Variations also exist in the behavior of the 50K-cleft, which apparently opens upon addition of ADP to the non-muscle isoforms but not to other isoforms. The fast-skeletal S1 remains as the only isoform that does not clearly exhibit either of the structural changes. The results indicate that the "conventional" myosin-II isoforms exhibit a wide variety of structural behavior, possibly depending on their functions and/or the history of molecular evolution.

PubMed Abstract


 
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