Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

The Work Performed By Biological Motors

Molecular motors are enzymes that perform work (F · x) when they move along a track a distance x against a constant force F. This work is performed through intermediate chemical steps in a motor’s ATPase reaction cycle, each step having a free energy change associated with it that is a sum of chemical, Δµchem, and mechanical, Δµext, potentials. Defining Δµext is fundamental to our understanding of how molecular motors work, yet after decades of study the definition of Δµext remains disputed. Some postulate that Δµext is a function of both F and x, while others assume that Δµext is a function of neither F nor x, and still others argue that Δµext is a function of F but not x. Here we evaluate these models and conclude that only the latter – a mechanochemical model proposed by A.V. Hill in the 1930’s – describes molecular motor mechanochemistry.

Mg 2+ -dependent ATPase activity in triatomine salivary glands (Heteroptera, Triatominae)

ABSTRACT Mg2+-ATPase activity was detected in the three salivary glands of adult triatomines, males and females, of Triatoma infestans (Klug, 1834) and Panstrongylus megistus (Burmeister, 1835) (Heteroptera, Triatominae). A predominance of binucleated cells in D1 and D2 and mononucleated in D3 was observed, with bulky and polyploidy nuclei. ATPase activity was detected in the nuclei, possibly in euchromatin and nucleolus, where this enzyme probably acts in the transcription process. ATPase reaction was also evidenced in the nuclear membrane, which is probably associated with nuclear-cytoplasmatic transport. These characteristics indicate a high metabolism and protein synthesis, which must be essential to saliva production as well as in maintaining the hematophagy of triatomines.

RecBCD possesses strong coupling between DNA and nucleotide binding that may propel a stepping mechanism during translocation

Double strand breaks are the severest genomic damage requiring rapid repair response. In prokaryotes, members of the RecBCD family initiate DNA unwinding essential for double strand break repair mechanism by homologous recombination. RecBCD is a highly processive DNA helicase with an unwinding rate approaching ∼1,600 bp·s-1. The ATPase reaction mechanism enabling RecBCD to achieve this fast unwinding rate and its enzymatic adaptation are not fully understood. Here, we present thermodynamic investigation of DNA and nucleotide binding to RecBCD to reveal the binding linkage and the degree of coupling between its nucleotide cofactor and DNA substrate binding. We find that RecBCD exhibits a weak binding state in the presence of ADP towards double overhang DNA substrate (dohDNA), and the same degree of coupling is observed for RecBCD affinity toward ADP, only in the presence of dohDNA. In the absence of nucleotide cofactor (APO state) or in the presence of AMPpNp, much weaker coupling is observed between the binding of DNA and the nucleotide state towards RecBCD. Other DNA substrates that are not optimally engaged with RecBCD do not exhibit similar degree of coupling. This may be the first evidence for strong and weak binding states that can, in principle, regulate a ‘stepping mechanism’ during processive translocation of RecBCD.

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858)

ABSTRACTAAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of aMycobacterium smegmatisAAA protein Msm0858, the ortholog ofMycobacterium tuberculosisRv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two β-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer.IMPORTANCERecent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal β-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

The role of monovalent cations in the ATPase reaction of DNA gyrase

Four new crystal structures of the ATPase domain of the GyrB subunit ofEscherichia coliDNA gyrase have been determined. One of these, solved in the presence of K+, is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K+ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na+ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.

Activity of Na+/K+-activated Mg2+-dependent ATP-hydrolase in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9

The aim of our work was to study Na+/K+-activated Mg2+-dependent ATPase activity in cell-free extracts of the sulfate-reducing bacteriaDesulfovibrio pigerVib-7 andDesulfomicrobiumsp. Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme reaction. The maximum ATPase activity for both bacterial strains at +35 ºC was determined. The highest activities of the studied enzyme in the cell-free extracts ofD. pigerVib-7 at pH 7.0 andDesulfomicrobiumsp. Rod-9 at pH 6.5 were measured. Based on experimental data, the analysis of kinetic properties of the ATP-hydrolase reaction by the studied bacteria was carried out. The enzyme activity, initial (instantaneous) reaction rate (V0) and maximum rate of the ATPase reaction (Vmax) was significantly higher inD. pigerVib-7 cells than inDesulfomicrobiumsp. Rod-9. Michaelis constants (Km) of the enzyme reaction for both bacterial strains were determined.