Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle.

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

Proteasomes: unfoldase-assisted protein degradation machines.

Proteasomes are the principal molecular machines for the regulated degradation of intracellular proteins. These self-compartmentalized macromolecular assemblies selectively degrade misfolded, mistranslated, damaged or otherwise unwanted proteins, and play a pivotal role in the maintenance of cellular proteostasis, in stress response, and numerous other processes of vital importance. Whereas the molecular architecture of the proteasome core particle (CP) is universally conserved, the unfoldase modules vary in overall structure, subunit complexity, and regulatory principles. Proteasomal unfoldases are AAA+ ATPases (ATPases associated with a variety of cellular activities) that unfold protein substrates, and translocate them into the CP for degradation. In this review, we summarize the current state of knowledge about proteasome - unfoldase systems in bacteria, archaea, and eukaryotes, the three domains of life.

Recognition of chromatin by the plant alkaloid, ellipticine as a dual binder.

Recognition of core histone components of chromatin along with chromosomal DNA by a class of small molecule modulators is worth examining to evaluate their intracellular mode of action. A plant alkaloid ellipticine (ELP) which is a putative anticancer agent has so far been reported to function via DNA intercalation, association with topoisomerase II and binding to telomere region. However, its effect upon the potential intracellular target, chromatin is hitherto unreported. Here we have characterized the biomolecular recognition between ELP and different hierarchical levels of chromatin. The significant result is that in addition to DNA, it binds to core histone(s) and can be categorized as a 'dual binder'. As a sequel to binding with histone(s) and core octamer, it alters post-translational histone acetylation marks. We have further demonstrated that it has the potential to modulate gene expression thereby regulating several key biological processes such as nuclear organization, transcription, translation and histone modifications.

Mechanism of interaction of small transcription inhibitors with DNA in the context of chromatin and telomere.

Small molecules from natural and synthetic sources have long been employed as human drugs. The transcription inhibitory potential of one class of these molecules has paved their use as anticancer drugs. The principal mode of action of these molecules is via reversible interaction with genomic DNA, double and multiple stranded. In this article we have revisited the mechanism of the interaction in the context of chromatin and telomere. The established modes of association of these molecules with double helical DNA provide a preliminary mechanism of their transcription inhibitory potential, but the scenario assumes a different dimension when the genomic DNA is associated with proteins in the transcription apparatus of both prokaryotic and eukaryotic organisms. We have discussed this altered scenario as a prelude to understand the chemical biology of their action in the cell. For the telomeric quadruplex DNA, we have reviewed the mechanism of their association with the quadruplex and resultant cellular consequence.

Self-association of the anionic form of the DNA-binding anticancer drug mithramycin.

The aqueous-phase self-association of mithramycin (MTR), an aureolic acid anticancer antibiotic, has been studied using different spectroscopic techniques such as absorption, fluorescence, circular dichroism, and 1H nuclear magnetic resonance spectroscopy. Results from these studies indicate self-association of the anionic antibiotic at pH 8.0 over a concentration range from micromolar to millimolar. These results could be ascribed to the following steps of self-association: M + M left arrow over right arrow M2, M2 + M left arrow over right arrow M3, and M3 + M left arrow over right arrow M4, where M, M2, M3, and M4 represent the monomer, dimer, trimer, and tetramer of mithramycin, respectively. Dynamic light scattering and isothermal titration calorimetry studies also support aggregation. In contrast, an insignificant extent of self-association is found for the neutral drug (at pH 3.5) and the [(MTR)2Mg2+] complex (at pH 8.0). Analysis of 2D NMR spectra of 1 mM MTR suggests that the sugar moieties play a role in the self-association process. Self-association of the drug might occur either via hydrophobic interaction of the sugar residues among themselves or water-mediated hydrogen bond formation between sugar residue(s). On the other hand, absence of a significant upfield shift of the aromatic protons from 100 microM to 1 mM MTR suggests against the possibility of stacking interactions between the aromatic rings as a stabilizing force for the formation of the dimer and higher oligomers.

Chromatin as a target for the DNA-binding anticancer drugs.

Chemotherapy has been a major approach to treat cancer. Both constituents of chromatin, chromosomal DNA and the associated chromosomal histone proteins are the molecular targets of the anticancer drugs. Small DNA binding ligands, which inhibit enzymatic processes with DNA substrate, are well known in cancer chemotherapy. These drugs inhibit the polymerase and topoisomerase activity. With the advent in the knowledge of chromatin chemistry and biology, attempts have shifted from studies of the structural basis of the association of these drugs or small ligands (with the potential of drugs) with DNA to their association with chromatin and nucleosome. These drugs often inhibit the expression of specific genes leading to a series of biochemical events. An overview will be given about the latest understanding of the molecular basis of their action. We shall restrict to those drugs, synthetic or natural, whose prime cellular targets are so far known to be chromosomal DNA.

Ultrafast dynamics in a nanocage of enzymes: solvation and fluorescence resonance energy transfer in reverse micelles.

In this contribution we report studies of the nature of solvation and resonance energy transfer processes in a reverse micelle (RM) upon encapsulation of a digestive enzyme, alpha-chymotrypsin (CHT). We have used one donor, Coumarin 500 (C500), and three acceptors Rhodamine 123 (R123, cationic), ethidium bromide (EtBr, cationic), and Merocyanine 540 (MC540, anionic). By selectively exciting the donor at the surface of the RM with a proper excitation wavelength we have examined solvation dynamics in the microenvironment. The solvation correlation function in the RM without CHT exhibits single-exponential decay with time constant approximately 660 ps, which is similar to that of the CHT-included RM. However, in the case of CHT-included RM (w(0)=10), the time-resolved anisotropy and spectral linewidth analysis of the surface-bound donor reveal the existence of an annular aqueous channel of thickness approximately 2.5 A between the enzyme surface and the inner surface of the RM. The aqueous channel is a potential host for the water-soluble substrate and also is involved in maintaining the proper functionality of RM encapsulated CHT. The studies use both steady-state and time-resolved fluorescence resonance energy transfer (FRET) techniques to measure donor-acceptor distances in the RM and also emphasize the danger of using steady-state fluorescence quenching as a method in careful estimation of the distances. The local geometrical restriction on the donor and acceptor molecules was estimated from time-resolved polarization (anisotropy) measurements. The time-resolved anisotropy of the donor and acceptor molecules also revealed significant randomization of the relative orientation of transition dipoles of the donor and acceptor, justifying the use of 2/3 as the value of the orientation factor kappa2. These studies attempt to elucidate the excellence of the RM as a nanohost of biological macromolecules.

The DNA intercalators ethidium bromide and propidium iodide also bind to core histones.

Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.

Minor groove binder distamycin remodels chromatin but inhibits transcription.

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance.

A revisit of the mode of interaction of small transcription inhibitors with genomic DNA.

One class of small molecules with therapeutic potential for treatment of cancer functions as transcription inhibitors via interaction with double-stranded DNA. Majority of the studies of the interaction with DNA have so far been reported under conditions nonexistent in vivo. Inside the cell, DNA is present in the nucleus as a complex with proteins known as chromatin. For the last few years we have been studying the interaction of these DNA-binding small molecules at the chromatin level with emphasis on the drug-induced structural alterations in chromatin. Our studies have shown that at the chromatin level these molecules could be classified in two broad categories: single-binding and dual-binding molecules. Single-binding molecules access only DNA in the chromatin, while the dual-binding molecules could bind to both DNA and the associated histone(s). Structural effects of the DNA-binding molecules upon chromatin in light of the above broad categories and the associated biological implications of the two types of binding are discussed.

Effect of DNA groove binder distamycin A upon chromatin structure.

BACKGROUND: Distamycin A is a prototype minor groove binder, which binds to B-form DNA, preferentially at A/T rich sites. Extensive work in the past few decades has characterized the binding at the level of double stranded DNA. However, effect of the same on physiological DNA, i.e. DNA complexed in chromatin, has not been well studied. Here we elucidate from a structural perspective, the interaction of distamycin with soluble chromatin, isolated from Sprague-Dawley rat. METHODOLOGY/PRINCIPAL FINDINGS: Chromatin is a hierarchical assemblage of DNA and protein. Therefore, in order to characterize the interaction of the same with distamycin, we have classified the system into various levels, according to the requirements of the method adopted, and the information to be obtained. Isothermal titration calorimetry has been employed to characterize the binding at the levels of chromatin, chromatosome and chromosomal DNA. Thermodynamic parameters obtained thereof, identify enthalpy as the driving force for the association, with comparable binding affinity and free energy for chromatin and chromosomal DNA. Reaction enthalpies at different temperatures were utilized to evaluate the change in specific heat capacity (ΔCp), which, in turn, indicated a possible binding associated structural change. Ligand induced structural alterations have been monitored by two complementary methods--dynamic light scattering, and transmission electron microscopy. They indicate compaction of chromatin. Using transmission electron microscopy, we have visualized the effect of distamycin upon chromatin architecture at di- and trinucleosome levels. Our results elucidate the simultaneous involvement of linker bending and internucleosomal angle contraction in compaction process induced by distamycin. CONCLUSIONS/SIGNIFICANCE: We summarize here, for the first time, the thermodynamic parameters for the interaction of distamycin with soluble chromatin, and elucidate its effect on chromatin architecture. The study provides insight into a ligand induced compaction phenomenon, and suggests new mechanisms of chromatin architectural alteration.