Intrinsic structural dynamics dictate enzymatic activity and inhibition.

Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors.

Impact of Side-Chain Hydrophilicity on Packing, Swelling, and Ion Interactions in Oxy-Bithiophene Semiconductors.

Exchanging hydrophobic alkyl-based side chains to hydrophilic glycol-based side chains is a widely adopted method for improving mixed-transport device performance, despite the impact on solid-state packing and polymer-electrolyte interactions being poorly understood. Presented here is a molecular dynamics (MD) force field for modeling alkoxylated and glycolated polythiophenes. The force field is validated against known packing motifs for their monomer crystals. MD simulations, coupled with X-ray diffraction (XRD), show that alkoxylated polythiophenes will pack with a "tilted stack" and straight interdigitating side chains, whilst their glycolated counterpart will pack with a "deflected stack" and an s-bend side-chain configuration. MD simulations reveal water penetration pathways into the alkoxylated and glycolated crystals-through the π-stack and through the lamellar stack respectively. Finally, the two distinct ways triethylene glycol polymers can bind to cations are revealed, showing the formation of a metastable single bound state, or an energetically deep double bound state, both with a strong side-chain length dependence. The minimum energy pathways for the formation of the chelates are identified, showing the physical process through which cations can bind to one or two side chains of a glycolated polythiophene, with consequences for ion transport in bithiophene semiconductors.

Aromatic side-chain flips orchestrate the conformational sampling of functional loops in human histone deacetylase 8.

Human histone deacetylase 8 (HDAC8) is a key hydrolase in gene regulation and an important drug-target. High-resolution structures of HDAC8 in complex with substrates or inhibitors are available, which have provided insights into the bound state of HDAC8 and its function. Here, using long all-atom unbiased molecular dynamics simulations and Markov state modelling, we show a strong correlation between the conformation of aromatic side chains near the active site and opening and closing of the surrounding functional loops of HDAC8. We also investigated two mutants known to allosterically downregulate the enzymatic activity of HDAC8. Based on experimental data, we hypothesise that I19S-HDAC8 is unable to release the product, whereas both product release and substrate binding are impaired in the S39E-HDAC8 mutant. The presented results deliver detailed insights into the functional dynamics of HDAC8 and provide a mechanism for the substantial downregulation caused by allosteric mutations, including a disease causing one.

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. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.

Intra-residue methyl-methyl correlations for valine and leucine residues in large proteins from a 3D-HMBC-HMQC experiment.

Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a 'divide and conquer' approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

Determining isoleucine side-chain rotamer-sampling in proteins from 13C chemical shift.

Chemical shifts are often the only nuclear magnetic resonance parameter that can be obtained for challenging macromolecular systems. Here we present a framework to derive the conformational sampling of isoleucine side chains from 13C chemical shifts and demonstrate that side-chain conformations in a low-populated folding intermediate can be determined.