Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states.

Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure-activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation.

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases.

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase.

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α2ßß'ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPR and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.

A water-mediated allosteric network governs activation of Aurora kinase A.

The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium. Tpx2 further activates AurA by stabilizing a water-mediated allosteric network that links the C-helix to the active site through an unusual polar residue in the regulatory spine. The polar spine residue and water network of AurA are essential for phosphorylation-driven activation, but an alternative form of the water network found in related kinases can support Tpx2-driven activation, suggesting that variations in the water-mediated hydrogen bond network mediate regulatory diversification in protein kinases.

E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure.

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by >10(5)-fold and >10(2)-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ(70) region 1.1 (σ1.1), ß' jaw and ß' sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases -4 to +2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either ß' jaw or SI3 equalizes OC lifetimes for λPR and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.

Initial events in bacterial transcription initiation.

Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the -10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

Using solutes and kinetics to probe large conformational changes in the steps of transcription initiation.

Small solutes are useful probes of large conformational changes in RNA polymerase-promoter interactions and other biopolymer processes. In general, a large effect of a solute on an equilibrium constant (or rate constant) indicates a large change in water-accessible biopolymer surface area in the corresponding step (or transition state), resulting from conformational changes, interface formation, or both. Here, we describe nitrocellulose filter binding assays from series used to determine the urea dependence of open complex formation and dissociation with Escherichia coli RNA polymerase and phage λPR promoter DNA. Then, we describe the subsequent data analysis and interpretation of these solute effects.