Detection of Ligand-induced Conformational Changes in the Activation Loop of Aurora-A Kinase by PELDOR Spectroscopy.

The structure of protein kinases has been extensively studied by protein crystallography. Conformational movement of the kinase activation loop is thought to be crucial for regulation of activity; however, in many cases the position of the activation loop in solution is unknown. Protein kinases are an important class of therapeutic target and kinase inhibitors are classified by their effect on the activation loop. Here, we report the use of pulsed electron double resonance (PELDOR) and site-directed spin labeling to monitor conformational changes through the insertion of MTSL [S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1 H-pyrrol-3-yl)methyl methanesulfonothioate] on the dynamic activation loop and a stable site on the outer surface of the enzyme. The action of different ligands such as microtubule-associated protein (TPX2) and inhibitors could be discriminated as well as their ability to lock the activation loop in a fixed conformation. This study provides evidence for structural adaptations that could be used for drug design and a methodological approach that has potential to characterize inhibitors in development

Interpreting findings from Mendelian randomization using the MR-Egger method

Mendelian randomization-Egger (MR-Egger) is an analysis method for Mendelian randomization using summarized genetic data. MR-Egger consists of three parts: (1) a test for directional pleiotropy, (2) a test for a causal effect, and (3) an estimate of the causal effect. While conventional analysis methods for Mendelian randomization assume that all genetic variants satisfy the instrumental variable assumptions, the MR-Egger method is able to assess whether genetic variants have pleiotropic effects on the outcome that differ on average from zero (directional pleiotropy), as well as to provide a consistent estimate of the causal effect, under a weaker assumption-the InSIDE (INstrument Strength Independent of Direct Effect) assumption. In this paper, we provide a critical assessment of the MR-Egger method with regard to its implementation and interpretation. While the MR-Egger method is a worthwhile sensitivity analysis for detecting violations of the instrumental variable assumptions, there are several reasons why causal estimates from the MR-Egger method may be biased and have inflated Type 1 error rates in practice, including violations of the InSIDE assumption and the influence of outlying variants. The issues raised in this paper have potentially serious consequences for causal inferences from the MR-Egger approach. We give examples of scenarios in which the estimates from conventional Mendelian randomization methods and MR-Egger differ, and discuss how to interpret findings in such cases.Stephen Burgess is supported by Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 204623/Z/16/Z). Simon G. Thompson is supported by the British Heart Foundation (Grant Number CH/12/2/ 29428)

Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

The vast majority of clinically-approved protein kinase inhibitors target the ATP binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken, and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors

Optimisation of a nano-positioning stage for a Transverse Dynamic Force Microscope

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.This paper describes the optimisation of a nano-positioning stage for a Transverse Dynamic Force Microscope (TDFM). The nano-precision stage is required to move a specimen dish within a horizontal region of 1 μm × 1 μm and with a resolution of 0.3 nm. The design objective was to maximise positional accuracy during high speed actuation. This was achieved by minimising out-of-plane distortions and vibrations during actuation. Optimal performance was achieved through maximising out-of-plane stiffness through shape and material selection as well optimisation of the anchoring system. Several shape parameters were optimised including the shape of flexural beams and the shape of the dish holder. Physical prototype testing was an essential part of the design process to confirm the accuracy of modelling and also to reveal issues with manufacturing tolerances. An overall resonant frequency of 6 kHz was achieved allowing for a closed loop-control frequency of 1.73 kHz for precise horizontal motion control. This resonance represented a 12-fold increase from the original 500 Hz of a commercially available positioning stage. Experimental maximum out-of-plane distortions below the first resonance frequency were reduced from 0.3 μm for the first prototype to less than 0.05 μm for the final practical prototype

Real-time sliding mode observer scheme for shear force estimation in a transverse dynamic force microscope

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record.This paper describes a sliding mode observer scheme for estimation of the shear force affecting the cantilever in a Transverse Dynamic Force Microscope (TDFM). The vertically oriented cantilever is oscillated in proximity to the specimen under investigation. The amplitude of oscillation of the cantilever tip is affected by these shear forces. They are created by the ordered-water layer above the specimen. The oscillation amplitude is therefore a measure of distance between the tip and the surface of the specimen. Consequently, the estimation of the shear forces provides useful information about the specimen characteristics. For estimating the shear forces, an approximate finite dimensional model of the cantilever is created using the method of lines. This model is subsequently reduced for its model order. An unknown input sliding mode observer has been used to reconstruct the unknown shear forces using only tip position measurements and the cantilever excitation. This paper describes the development of the sliding mode scheme and presents experimental results from the TDFM set up at the Centre for Nanoscience and Quantum Information (NSQI) at Bristol University

Sensitivity Analyses for Robust Causal Inference from Mendelian Randomization Analyses with Multiple Genetic Variants.

Mendelian randomization investigations are becoming more powerful and simpler to perform, due to the increasing size and coverage of genome-wide association studies and the increasing availability of summarized data on genetic associations with risk factors and disease outcomes. However, when using multiple genetic variants from different gene regions in a Mendelian randomization analysis, it is highly implausible that all the genetic variants satisfy the instrumental variable assumptions. This means that a simple instrumental variable analysis alone should not be relied on to give a causal conclusion. In this article, we discuss a range of sensitivity analyses that will either support or question the validity of causal inference from a Mendelian randomization analysis with multiple genetic variants. We focus on sensitivity analyses of greatest practical relevance for ensuring robust causal inferences, and those that can be undertaken using summarized data. Aside from cases in which the justification of the instrumental variable assumptions is supported by strong biological understanding, a Mendelian randomization analysis in which no assessment of the robustness of the findings to violations of the instrumental variable assumptions has been made should be viewed as speculative and incomplete. In particular, Mendelian randomization investigations with large numbers of genetic variants without such sensitivity analyses should be treated with skepticism.Stephen Burgess is funded by a fellowship from the Wellcome Trust (100114). Jack Bowden is supported by a Methodology Research Fellowship from the UK Medical Research Council (grant number MR/N501906/1). Simon G. Thompson is supported by the British Heart Foundation (grant number CH/12/2/29428)

Solution NMR assignment of the C-terminal domain of human chTOG

The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the 1H–15N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain

Effective theories of single field inflation when heavy fields matter

We compute the low energy effective field theory (EFT) expansion for single-field inflationary models that descend from a parent theory containing multiple other scalar fields. By assuming that all other degrees of freedom in the parent theory are sufficiently massive relative to the inflaton, it is possible to derive an EFT valid to arbitrary order in perturbations, provided certain generalized adiabaticity conditions are respected. These conditions permit a consistent low energy EFT description even when the inflaton deviates off its adiabatic minimum along its slowly rolling trajectory. By generalizing the formalism that identifies the adiabatic mode with the Goldstone boson of this spontaneously broken time translational symmetry prior to the integration of the heavy fields, we show that this invariance of the parent theory dictates the entire non-perturbative structure of the descendent EFT. The couplings of this theory can be written entirely in terms of the reduced speed of sound of adiabatic perturbations. The resulting operator expansion is distinguishable from that of other scenarios, such as standard single inflation or DBI inflation. In particular, we re-derive how certain operators can become transiently strongly coupled along the inflaton trajectory, consistent with slow-roll and the validity of the EFT expansion, imprinting features in the primordial power spectrum, and we deduce the relevant cubic operators that imply distinct signatures in the primordial bispectrum which may soon be constrained by observations.Comment: (v1) 25 pages, 1 figure; (v2) references added and typos corrected, to appear in Journal of High Energy Physic

Density functional theory studies of MTSL nitroxide side chain conformations attached to an activation loop

A quantum-mechanical (QM) method rooted on density functional theory (DFT) linked to the Stochastic Liouville equation (SLE) in the Fokker Planck (FP) form has been employed for the first time to sample the methane-thiosulfonate spin label (MTSL) conformational space attached to the Aurora-A kinase activation loop and to calculate the EPR spectrum. The features of the calculated energy surface allowed us to describe the system in a limited number of rotamers stabilized by interactions of the MTSL side chain and neighbouring residues. The relevant magnetic parameters and the electron paramagnetic resonance (EPR) spectrum were subsequently calculated from the trajectories of the spin probe in the protein environment. The comparison between theoretical and experimental continuous wave (CW) EPR spectra revealed some small differences in the EPR line shape which arises from the combinations of g- and A-values obtained from the conformations selected. The theoretical approach adopted in this work can be used to recognise the contribution of MTSL rotamers to the EPR spectrum in order to help extract structural/dynamics properties of protein from the experimental data

Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN–MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems