A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time-Resolved Luminescence Studies.

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

A radical sense of direction: signalling and mechanism in cryptochrome magnetoreception.

The remarkable phenomenon of magnetoreception in migratory birds and other organisms has fascinated biologists for decades. Much evidence has accumulated to suggest that birds sense the magnetic field of the Earth using photochemical transformations in cryptochrome flavoproteins. In the last 5 years this highly interdisciplinary field has seen advances in structural biology, biophysics, spin chemistry, and genetic studies in model organisms. We review these developments and consider how this chemical signal can be integrated into the cellular response.

Dynamic Equilibrium of the Aurora†A Kinase Activation Loop Revealed by Single-Molecule Spectroscopy.

The conformation of the activation loop (T-loop) of protein kinases underlies enzymatic activity and influences the binding of small-molecule inhibitors. By using single-molecule fluorescence spectroscopy, we have determined that phosphorylated Aurora A kinase is in dynamic equilibrium between a DFG-in-like active T-loop conformation and a DFG-out-like inactive conformation, and have measured the rate constants of interconversion. Addition of the Aurora A activating protein TPX2 shifts the equilibrium towards an active T-loop conformation whereas addition of the inhibitors MLN8054 and CD532 favors an inactive T-loop. We show that Aurora A binds TPX2 and MLN8054 simultaneously and provide a new model for kinase conformational behavior. Our approach will enable conformation-specific effects to be integrated into inhibitor discovery across the kinome, and we outline some immediate consequences for structure-based drug discovery.

Sensitive fluorescence-based detection of magnetic field effects in photoreactions of flavins.

Magnetic field effect studies have been conducted on a variety of flavin-based radical pair systems chosen to model the magnetosensitivity of the photoinduced radical pairs found in cryptochrome flavoproteins. Cryptochromes are blue-light photoreceptor proteins which are thought to mediate avian magnetoreception, an hypothesis supported by recent in vitro observations of magnetic field-dependent reaction kinetics for a light-induced radical pair in a cryptochrome from the plant Arabidopsis thaliana. Many cryptochromes are difficult to express in large quantities or high concentrations and are easily photodegraded. Magnetic field effects are typically measured by spectroscopic detection of the transient radical (pair) concentrations. Due to its low sensitivity, single-pass transient absorption spectroscopy can be of limited use in such experiments and much recent work has involved development of other methodologies offering improved sensitivity. Here we explore the use of flavin fluorescence as the magnetosensitive probe and demonstrate the exceptional sensitivity of this technique which allows the detection of magnetic field effects in flavin samples at sub-nanomolar concentrations and in cryptochromes.

Activation of Aurora-A kinase by protein partner binding and phosphorylation are independent and synergistic.

Protein kinases are activated by phosphorylation and by the binding of activator proteins. The interplay of these two factors is incompletely understood. We applied energetic analysis to this question and characterized the activation process of the serine/threonine kinase Aurora-A by phosphorylation and by its protein partner, targeting protein for Xenopus kinesin-like protein 2 (TPX2). We discovered that these two activators act synergistically and without a predefined order: each can individually increase the activity of Aurora-A, and the effect of both bound together is the exact sum of their individual contributions to catalysis. Unexpectedly, the unphosphorylated enzyme has catalytic activity that is increased 15-fold by the binding of TPX2 alone. The energetic contribution of phosphorylation to catalysis is 2-fold greater than that of TPX2 binding, which is independent of the phosphorylation state of the enzyme. Based on this analysis, we propose a revised, fluid model of Aurora-A activation in which the first step is a reduction in the mobility of the activation loop by either TPX2 binding or phosphorylation. Furthermore, our results suggest that unphosphorylated Aurora-A bound to the mitotic spindle by TPX2 is catalytically active and that the phosphorylation state of Aurora-A is an inaccurate surrogate for its activity. Extending this form of analysis will allow us to compare quantitatively the effects of the whole network of kinase-activating partners. Comparison with other kinases showed that kinetic characterization detects those kinases whose activation loops undergo a rearrangement upon phosphorylation and thus whose unphosphorylated state offers a distinct target for the development of Type II inhibitors.

The structural mechanisms that underpin mitotic kinase activation.

In eukaryotic cells, the peak of protein phosphorylation occurs during mitosis, switching the activities of a significant proportion of proteins and orchestrating a wholesale reorganization of cell shape and internal architecture. Most mitotic protein phosphorylation events are catalysed by a small subset of serine/threonine protein kinases. These include members of the Cdk (cyclin-dependent kinase), Plk (Polo-like kinase), Aurora, Nek (NimA-related kinase) and Bub families, as well as Haspin, Greatwall and Mps1/TTK. There has been steady progress in resolving the structural mechanisms that regulate the catalytic activities of these mitotic kinases. From structural and biochemical perspectives, kinase activation appears not as a binary process (from inactive to active), but as a series of states that exhibit varying degrees of activity. In its lowest activity state, a mitotic kinase may exhibit diverse autoinhibited or inactive conformations. Kinase activation proceeds via phosphorylation and/or association with a binding partner. These remodel the structure into an active conformation that is common to almost all protein kinases. However, all mitotic kinases of known structure have divergent features, many of which are key to understanding their specific regulatory mechanisms. Finally, mitotic kinases are an important class of drug target, and their structural characterization has facilitated the rational design of chemical inhibitors.

Staphylococcal Complement Evasion Protein Sbi Stabilises C3d Dimers by Inducing an N-Terminal Helix Swap.

Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.

Crystal structure of an Aurora-A mutant that mimics Aurora-B bound to MLN8054: insights into selectivity and drug design.

The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple-point mutant of Aurora-A (AurAx3) which mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3 in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the T217E substitution, which we have confirmed using a single-point mutant. The activation loop of Aurora-A in the AurAx3-MLN8054 complex exhibits an unusual conformation in which Asp274 and Phe275 side chains point into the interior of the protein. There is to our knowledge no documented precedent for this conformation, which we have termed DFG-up. The sequence requirements of the DFG-up conformation suggest that it might be accessible to only a fraction of kinases. MLN8054 thus circumvents the problem of highly homologous active sites. Binding of MLN8054 to Aurora-A switches the character of a pocket within the active site from polar to a hydrophobic pocket, similar to what is observed in the structure of Aurora-A bound to a compound that induces DFG-out. We propose that targeting this pocket may be a productive route in the design of selective kinase inhibitors and describe the structural basis for the rational design of these compounds.

Ligand discrimination between active and inactive activation loop conformations of Aurora-A kinase is unmodified by phosphorylation.

Structure-based drug design is commonly used to guide the development of potent and specific enzyme inhibitors. Many enzymes - such as protein kinases - adopt multiple conformations, and conformational interconversion is expected to impact on the design of small molecule inhibitors. We measured the dynamic equilibrium between DFG-in-like active and DFG-out-like inactive conformations of the activation loop of unphosphorylated Aurora-A alone, in the presence of the activator TPX2, and in the presence of kinase inhibitors. The unphosphorylated kinase had a shorter residence time of the activation loop in the active conformation and a shift in the position of equilibrium towards the inactive conformation compared with phosphorylated kinase for all conditions measured. Ligand binding was associated with a change in the position of conformational equilibrium which was specific to each ligand and independent of the kinase phosphorylation state. As a consequence of this, the ability of a ligand to discriminate between active and inactive activation loop conformations was also independent of phosphorylation. Importantly, we discovered that the presence of multiple enzyme conformations can lead to a plateau in the overall ligand K d, despite increasing affinity for the chosen target conformation, and modelled the conformational discrimination necessary for a conformation-promoting ligand.

A simple, robust, universal assay for real-time enzyme monitoring by signalling changes in nucleoside phosphate anion concentration using a europium(iii)-based anion receptor.

Enzymes that consume and produce nucleoside polyphosphate (NPP) anions represent major targets in drug discovery. For example, protein kinases are one of the largest classes of drug targets in the fight against cancer. The accurate determination of enzyme kinetics and mechanisms is a critical aspect of drug discovery research. To increase confidence in the selection of lead drug compounds it is crucial that pharmaceutical researchers have robust, affordable assays to measure enzyme activity accurately. We present a simple, sensitive microplate assay for real-time monitoring of a range of pharmaceutically important enzyme reactions that generate NPP anions, including kinases and glycosyltransferases. Our assay utilises a single, stable europium(iii) complex that binds reversibly to NPP anions, signalling the dynamic changes in NPP product/substrate ratio during an enzyme reaction using time-resolved luminescence. This supramolecular approach to enzyme monitoring overcomes significant limitations in existing assays, obviating the need for expensive antibodies or equipment, chemically labelled substrates or products and isolation or purification steps. Our label and antibody-free method enables rapid and quantitative analysis of enzyme activities and inhibition, offering a potentially powerful tool for use in drug discovery, suitable for high-throughput screening of inhibitors and accurate measurements of enzyme kinetic parameters.

Production of Protein Kinases in E. coli.

Recombinant protein expression is widely used to generate milligram quantities of protein kinases for crystallographic, enzymatic, or other biophysical assays in vitro. Expression in E. coli is fast, cheap, and reliable. Here I present a detailed protocol for the production of human Aurora-A kinase. I begin with transformation of a suitable plasmid into an expression strain of E. coli, followed by growth and harvesting of bacterial cell cultures. Finally, I describe the purification of Aurora-A to homogeneity using immobilized metal affinity and size exclusion chromatographies.

Kinetic Analysis Reveals the Identity of Aß-Metal Complex Responsible for the Initial Aggregation of Aß in the Synapse.

The mechanism of Aß aggregation in the absence of metal ions is well established, yet the role that Zn2+ and Cu2+, the two most studied metal ions, released during neurotransmission, paly in promoting Aß aggregation in the vicinity of neuronal synapses remains elusive. Here we report the kinetics of Zn2+ binding to Aß and Zn2+/Cu2+ binding to Aß-Cu to form ternary complexes under near physiological conditions (nM Aß, µM metal ions). We find that these reactions are several orders of magnitude slower than Cu2+ binding to Aß. Coupled reaction-diffusion simulations of the interactions of synaptically released metal ions with Aß show that up to a third of Aß is Cu2+-bound under repetitive metal ion release, while any other Aß-metal complexes (including Aß-Zn) are insignificant. We therefore conclude that Zn2+ is unlikely to play an important role in the very early stages (i.e., dimer formation) of Aß aggregation, contrary to a widely held view in the subject. We propose that targeting the specific interactions between Cu2+ and Aß may be a viable option in drug development efforts for early stages of AD.

Chemical amplification of magnetic field effects relevant to avian magnetoreception.

Magnetic fields as weak as the Earth's can change the yields of radical pair reactions even though the energies involved are orders of magnitude smaller than the thermal energy, kBT, at room temperature. Proposed as the source of the light-dependent magnetic compass in migratory birds, the radical pair mechanism is thought to operate in cryptochrome flavoproteins in the retina. Here we demonstrate that the primary magnetic field effect on flavin photoreactions can be amplified chemically by slow radical termination reactions under conditions of continuous photoexcitation. The nature and origin of the amplification are revealed by studies of the intermolecular flavin-tryptophan and flavin-ascorbic acid photocycles and the closely related intramolecular flavin-tryptophan radical pair in cryptochrome. Amplification factors of up to 5.6 were observed for magnetic fields weaker than 1 mT. Substantial chemical amplification could have a significant impact on the viability of a cryptochrome-based magnetic compass sensor.

Protein folding of the SAP domain, a naturally occurring two-helix bundle.

The SAP domain from the Saccharomyces cerevisiae Tho1 protein is comprised of just two helices and a hydrophobic core and is one of the smallest proteins whose folding has been characterised. Φ-value analysis revealed that Tho1 SAP folds through a transition state where helix 1 is the most extensively formed element of secondary structure and flickering native-like core contacts from Leu35 are also present. The contacts that contribute most to native state stability of Tho1 SAP are not formed in the transition state.

A kinetic test characterizes kinase intramolecular and intermolecular autophosphorylation mechanisms.

Many protein kinases catalyze their own activation by autophosphorylation. The mechanism of this is generally considered to be intermolecular and similar to that used in substrate phosphorylation. We derived the kinetic signatures of the four simplest autophosphorylation reactions and developed a test to determine the autoactivation mechanism of individual kinases. Whereas autophosphorylation of Nek7 and Plk4 occurred through an intermolecular mechanism, the kinases Aurora-A and Chk2 followed an intramolecular mechanism. Autophosphorylation of Aurora-A was accelerated in the presence of its protein activator TPX2. Nek9, the binding partner for Nek7, had a concentration-dependent effect such that low amounts enhanced autoactivation of Nek7 and high amounts were inhibitory. A structural model of Aurora-A undergoing autophosphorylation confirmed that an intramolecular mechanism is physically possible, and provided an explanation for how TPX2 could stimulate both autophosphorylation and substrate phosphorylation. The distinct mechanisms of autoactivation have consequences for cellular regulation because each molecule of a kinase that undergoes intramolecular autophosphorylation is activated individually, whereas the activity of kinases that undergo intermolecular autophosphorylation can be rapidly self-amplified in the cell. Local control of individual molecules, such as Aurora-A, may be particularly advantageous for a kinase with multiple, distinct cellular roles.

Magnetic field effects in flavoproteins and related systems.

Within the framework of the radical pair mechanism, magnetic fields may alter the rate and yields of chemical reactions involving spin-correlated radical pairs as intermediates. Such effects have been studied in detail in a variety of chemical systems both experimentally and theoretically. In recent years, there has been growing interest in whether such magnetic field effects (MFEs) also occur in biological systems, a question driven most notably by the increasing body of evidence for the involvement of such effects in the magnetic compass sense of animals. The blue-light photoreceptor cryptochrome is placed at the centre of this debate and photoexcitation of its bound flavin cofactor has indeed been shown to result in the formation of radical pairs. Here, we review studies of MFEs on free flavins in model systems as well as in blue-light photoreceptor proteins and discuss the properties that are crucial in determining the magnetosensitivity of these systems.

Engineering a two-helix bundle protein for folding studies.

The SAP domain from the Saccharomyces cerevisiae THO1 protein contains a hydrophobic core and just two alpha-helices. It could provide a system for studying protein folding that bridges the gap between studies on isolated helices and those on larger protein domains. We have engineered the SAP domain for protein folding studies by inserting a tryptophan residue into the hydrophobic core (L31W) and solved its structure. The helical regions had a backbone root mean-squared deviation of 0.9 A from those of wild type. The mutation L31W destabilised wild type by 0.8 +/- 0.1 kcal mol(-1). The mutant folded in a reversible, apparent two-state manner with a microscopic folding rate constant of around 3700 s(-1) and is suitable for extended studies of folding.