A distal regulatory region of a class I human histone deacetylase.

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states.

Characterization of the Menin-MLL Interaction as Therapeutic Cancer Target.

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes.

Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Heteronuclear transverse and longitudinal relaxation in AX4 spin systems: application to (15)N relaxations in (15)NH4(+).

The equations that describe the time-evolution of transverse and longitudinal (15)N magnetisations in tetrahedral ammonium ions, (15)NH4(+), are derived from the Bloch-Wangsness-Redfield density operator relaxation theory. It is assumed that the relaxation of the spin-states is dominated by (1) the intra-molecular (15)N-(1)H and (1)H-(1)H dipole-dipole interactions and (2) interactions of the ammonium protons with remote spins, which also include the contribution to the relaxations that arise from the exchange of the ammonium protons with the bulk solvent. The dipole-dipole cross-correlated relaxation mechanisms between each of the (15)N-(1)H and (1)H-(1)H interactions are explicitly taken into account in the derivations.

Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor.

ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically.

Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.

A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to characterise potassium binding sites in enzymes: the (1) H,(15) N spin-pair of enzyme-bound (15) NH4 (+) can be probed by (15) N-edited heteronuclear NMR experiments.

Loop interactions and dynamics tune the enzymatic activity of the human histone deacetylase 8.

The human histone deacetylase 8 (HDAC8) is a key hydrolase in gene regulation and has been identified as a drug target for the treatment of several cancers. Previously the HDAC8 enzyme has been extensively studied using biochemical techniques, X-ray crystallography, and computational methods. Those investigations have yielded detailed information about the active site and have demonstrated that the substrate entrance surface is highly dynamic.

The molecular mechanism of Hsp100 chaperone inhibition by the prion curing agent guanidinium chloride.

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance.

Coupling of oligomerization and nucleotide binding in the AAA+ chaperone ClpB.

Members of the family of ATPases associated with various cellular activities (AAA+) typically form homohexameric ring complexes and are able to remodel their substrates, such as misfolded proteins or protein-protein complexes, in an ATP-driven process. The molecular mechanism by which ATP hydrolysis is coordinated within the multimeric complex and the energy is converted into molecular motions, however, is poorly understood. This is partly due to the fact that the oligomers formed by AAA+ proteins represent a highly complex system and analysis depends on simplification and prior knowledge.

Disaggregases in 4 dimensions.

Non-destructive dissagregation of protein aggregates is a formidable task mediated by the specialized AAA+ chaperone Hsp104/ClpB in combination with the Hsp70/DnaK chaperone system. The exact mechanism of how the hexameric Hsp104/ClpB proteins perform the task of protein disaggregation or remodeling is largely unknown. The process is ATP-dependent and tight coupling between the ATPase domains within the hexameric ring-complex could be observed.

Nucleotide binding and allosteric modulation of the second AAA+ domain of ClpB probed by transient kinetic studies.

The bacterial AAA+ chaperone ClpB provides thermotolerance by disaggregating aggregated proteins in collaboration with the DnaK chaperone system. Like many other AAA+ proteins, ClpB is believed to act as a biological motor converting the chemical energy of ATP into molecular motion. ClpB has two ATPase domains, NBD1 and NBD2, on one polypeptide chain.

Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c3.

Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking.

Shifting transition states in the unfolding of a large ankyrin repeat protein.

The 33-amino-acid ankyrin motif comprises a beta-turn followed by two anti-parallel alpha-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded.

Coupling and dynamics of subunits in the hexameric AAA+ chaperone ClpB.

The bacterial AAA+ protein ClpB and its eukaryotic homologue Hsp104 ensure thermotolerance of their respective organisms by reactivating aggregated proteins in cooperation with the Hsp70/Hsp40 chaperone system. Like many members of the AAA+ superfamily, the ClpB protomers form ringlike homohexameric complexes. The mechanical energy necessary to disentangle protein aggregates is provided by ATP hydrolysis at the two nucleotide-binding domains of each monomer.

The ATPase cycle of the mitochondrial Hsp90 analog Trap1.

Hsp90 is an ATP-dependent molecular chaperone whose mechanism is not yet understood in detail. Here, we present the first ATPase cycle for the mitochondrial member of the Hsp90 family called Trap1 (tumor necrosis factor receptor-associated protein 1). Using biochemical, thermodynamic, and rapid kinetic methods we dissected the kinetics of the nucleotide-regulated rearrangements between the open and the closed conformations.

Probing a moving target with a plastic unfolding intermediate of an ankyrin-repeat protein.

Repeat proteins are composed of tandem arrays of 30- to 40-residue structural motifs and are characterized by short-range interactions between residues close in sequence. Here we have investigated the equilibrium unfolding of D34, a 426-residue fragment of ankyrinR that comprises 12 ankyrin repeats. We show that D34 unfolds via an intermediate in which the C-terminal half of the protein is structured and the N-terminal half is unstructured.