SummaryCells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.

The binding of prolyl-hydroxylated HIF-α to PHD2 is hindered by prior 2OG binding; likely, leading to the inhibition of HIF-α degradation under limiting 2OG conditions.

Drug development for the treatment of central nervous system (CNS) diseases is extremely challenging, in large part due to the difficulty in crossing the blood-brain barrier (BBB). Here we develop and experimentally validate a new in silico method to predict quantitatively the BBB permeability for small-molecule drugs. We show accurate prediction of solute permeabilities at physiological temperature using high-temperature unbiased atomic detail molecular dynamics simulations of spontaneous drug diffusion across BBB bilayers. These simulations provide atomic detail insights into the transport mechanisms, as well as converged kinetics and thermodynamics. The method is validated computationally against physiological temperature simulations for fast-diffusing compounds, as well as experimentally by direct determination of the compound permeabilities using a transwell assay as an in vitro BBB model. The overall agreement of the predicted values with both direct simulations at physiological temperatures and experimental data is excellent. This new tool has the potential to replace current semi-empirical in silico screening and in vitro permeability measurements in CNS drug discovery.

The class D (OXA) serine β-lactamases are a major cause of resistance to β-lactam antibiotics. The class D enzymes are unique amongst β-lactamases because they have a carbamylated lysine that acts as a general acid/base in catalysis. Previous crystallographic studies led to the proposal that β-lactamase inhibitor avibactam targets OXA enzymes in part by promoting decarbamylation. Similarly, halide ions are proposed to inhibit OXA enzymes via decarbamylation. NMR analyses, in which the carbamylated lysines of OXA-10, -23 and -48 were C-labelled, indicate that reaction with avibactam does not ablate lysine carbamylation in solution. While halide ions did not decarbamylate theC-labelled OXA enzymes in the absence of substrate or inhibitor, avibactam-treated OXA enzymes were susceptible to decarbamylation mediated by halide ions, suggesting halide ions may inhibit OXA enzymes by promoting decarbamylation of acyl-enzyme complex. Crystal structures of the OXA-10 avibactam complex were obtained with bromide, iodide, and sodium ions bound between Trp-154 and Lys-70. Structures were also obtained wherein bromide and iodide ions occupy the position expected for the 'hydrolytic water' molecule. In contrast with some solution studies, Lys-70 was decarbamylated in these structures. These results reveal clear differences between crystallographic and solution studies on the interaction of class D β-lactamases with avibactam and halides, and demonstrate the utility of C-NMR for studying lysine carbamylation in solution.

Potassium channels are of paramount physiological and pathological importance and therefore constitute significant drug targets. One of the keys to rationalize the way drugs modulate ion channels is to understand the ability of such small molecules to access their respective binding sites, from which they can exert an activating or inhibitory effect. Many computational studies have probed the energetics of ion permeation, and the mechanisms of voltage gating, but little is known about the role of fenestrations as possible mediators of drug entry in potassium channels. To explore the existence, structure, and conformational dynamics of transmembrane fenestrations accessible by drugs in potassium channels, molecular dynamics simulation trajectories were analyzed from three potassium channels: the open state voltage-gated channel Kv1.2, the G protein-gated inward rectifying channel GIRK2 (Kir3.2), and the human two-pore domain TWIK-1 (K2P1.1). The main results of this work were the identification of the sequence identity of four main lateral fenestrations of similar length and with bottleneck radius in the range of 0.9-2.4 Å for this set of potassium channels. It was found that the fenestrations in Kv1.2 and Kir3.2 remain closed to the passage of molecules larger than water. In contrast, in the TWIK-1 channel, both open and closed fenestrations are sampled throughout the simulation, with bottleneck radius shown to correlate with the random entry of lipid membrane molecules into the aperture of the fenestrations. Druggability scoring function analysis of the fenestration regions suggests that Kv and Kir channels studied are not druggable in practice due to steric constraining of the fenestration bottleneck. A high (>50%) fenestration sequence identity was found in each potassium channel subfamily studied, Kv1, Kir3, and K2P1. Finally, the reported fenestration sequence of TWIK-1 compared favorably with another channel, K2P channel TREK-2, reported to possess open fenestrations, suggesting that K2P channels could be druggable via fenestrations, for which we reported atomistic detail of the fenestration region, including the flexible residues M260 and L264 that interact with POPC membrane in a concerted fashion with the aperture and closure of the fenestrations.

The chronic response of animals to hypoxia is mediated by the αβ-heterodimeric hypoxiainducible transcription factors (-HIFs) which upregulate the expression of sets of genes that work to ameliorate the effects of limiting dioxygen. The HIF prolyl hydroxylase domain enzymes (PHDs) are Fe(II) and 2-oxoglutarate dependent oxygenases that act as hypoxia sensing components of the HIF system: prolyl-hydroxylation signals for dioxgen availability dependent HIF-α degradation via the ubiquitin proteasome system. The unusual kinetic properties of the PHDs, in particular a high Km for dioxygen and slow reaction with dioxygen are proposed to enable their hypoxia sensing role. An understanding of how dioxygen is delivered to, and binds at, the active site of the PHDs is important for the development of a chemical understanding of the hypoxic response. We employed a combined multiscale approach involving classical atomistic equilibrium and non-equilibrium MD simulations combined with QM/MM trajectories to investigate dioxygen diffusion to, and binding at, the active site in the PHD2.Fe(II).2OG.HIF substrate complex; PHD2 is the most important of the three human PHDs. The transport of dioxygen to the active site is described; dioxygen transport follows a single well-defined hydrophobic tunnel, formed from both enzyme and substrate elements to reach the PHD2 active site. The results provide estimates for rate constants that define a diffusion-reaction model for dioxygen:PHD2 interactions; in combination with reported biophysical analyses they provide chemical insight into the basis of the slow reaction of PHD2 with dioxygen. They imply that the reversible binding of dioxygen is central to the hypoxia sensing capacity of the PHDs and that different PHD HIF-α substrate combinations might have different dioxygen sensitivity profiles.The extent of HIF-α substrate prolyl hydroxylation, which signals for subsequent HIF-α degradation is thus a manifestation of the equilibrium between dioxygen in bulk solution and dioxygen bound to the PHD2.Fe.2OG.HIF- substrate complex.

Voltage-gated potassium channels of the Kv1 family play a crucial role in the generation and transmission of electrical signals in excitable cells affecting neuronal and cardiac activities. Small-molecule blockage of these channels has been proposed to occur via a cooperative mechanism involving two main blocking sites: the inner-pore site located below the selectivity filter, and a side-pocket cavity located between the pore and the voltage sensor. Using 0.5 μs molecular dynamics simulation trajectories complemented by docking calculations, the potential binding sites of the PAP-1 (5-(4-phenoxybutoxy)psoralen) blocker to the crystal structure of Kv1.2 channel have been studied. The presence of both mentioned blocking sites at Kv1.2 is confirmed, adding evidence in favor of a cooperative channel blockage mechanism. These observations provide insight into drug modulation that will guide further developments of Kv inhibitors.

Collagen is the single most abundant protein in the extracellular matrix in the animal kingdom, with remarkable structural and functional diversity and regarded one of the most useful biomaterials. Etymologically, the term collagen comes from Greek kola 'glue' and gen 'giving birth to'. Thus, it is not surprising that the various collagens and the structures they form all serve the same purpose, to help tissues withstand stretching. Among the functions the various collagens are involved in are cell adhesion and migration, tissue repair, scaffolding and morphogenesis. Thus knowledge about the structure and properties of collagen, how they change depending on the nature of the local environment as well as the nature and specificity of collagen interactions with its partners is central to discerning the role of collagen in medical applications such as imaging, drug delivery and tissue engineering, and in the design and construction of synthetic collagen-like materials for tools in biomaterial science and nanotechnology. The main focus of this perspective is to review the molecular and packing structures of collagen and the computer simulations work performed up to now to further highlight the significance of collagen.