Supplementary guidance: listening to staff: Autumn 2017

Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance. Bacterial expression systems are superior to insect or mammalian cells in terms of simplicity and cost effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression produced high yields of Src and Abl kinase domains in bacteria, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 μg/mL of culture in an automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases and lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identifying kinase domain constructs previously expressed in bacteria and likely to express well in our protocol, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. Using a fluorescence-based thermostability assay and a fluorescent ATP-competitive inhibitor, we show that the highest-expressing kinases are folded and have well-formed ATP binding sites. We also demonstrate that these constructs can enable characterization of clinical mutations by expressing a panel of 48 Src and 46 Abl mutations. The wild-type kinase construct library is available publicly via Addgene

Identification of two different isomers of bosutinib.

<p>A) View of the ligand from our initial structure of Abl bound to the bosutinib isomer. The ligand is shown as sticks, colored according to the temperature factors (B-factors) of the atoms, with blue indicating low B-factors and red indicating high B-factors. The 2F_o-F_c electron density map, calculated with phases derived from a refined molecular model that included the 2-chloro group of bosutinib, is shown as a blue mesh. B) ¹H NMR spectra of bosutinib (Tocris Bioscience, blue) and the bosutinib isomer (LC Labs, red), showing only the aromatic region. C) View of the ligand from our structure of Abl bound to authentic bosutinib. The coordinates of bosutinib are shown as blue sticks. A simulated annealing omit map contoured at 0.8 standard deviations above the mean (0.8σ) is shown as a blue mesh. An anomalous difference map, contoured at 3.0σ, is shown in red. D) Fluorescence emission spectra (excitation at 280 nm) of 50 nM Abl kinase domain in the presence of varying concentrations of bosutinib (the spectra are colored according to bosutinib concentration, which was varied in 10 nM increments from 0 nM shown in blue to 60 nM shown in red). The inset shows a binding curve measured for 5 nM Abl. The normalized fluorescence intensity at 480 nm is plotted as a function of the total bosutinib concentration. The smooth line shows the numerical fit (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029828#s2" target="_blank">Materials and Methods</a>).</p

Bosutinib binds to both DFG-In and DFG-Out Abl.

<p>A) Comparison of the conformation of the activation loop and DFG motif in our structure (DFG-Out, yellow) and in the dasatinib cocrystal structure (DFG-In, gray). The sidechains of D381 in the dasatinib structure and F382 in our structure, which occupy very similar positions, are shown as spheres. Bosutinib is shown as sticks and spheres. The position of the phosphate group on the phosphorylated sidechain of Y393 in the dasatinib structure is shown as an orange sphere. B) Binding curves for bosutinib binding to Abl and to Abl phosphorylated on the activation loop (Abl-pY393).</p

The nitrile group of bosutinib and the bosutinib isomer probe electrostatics in the ATP-binding site.

<p>A) Infrared absorbance (top) and Stark (bottom) spectra of 50 mM bosutinib in 1-propanol, measured at 77 K. A numerical fit to the Stark spectrum, from which the linear Stark tuning rate was derived, is shown in red. The numerical fit is a weighted sum of the derivatives of the absorption spectrum, and the individual fit components are shown as thin lines. B) The nitrile stretch region of infrared absorbance spectra of bosutinib bound to the kinase domains of Abl (black), Src (red) and the Src T338I mutant (blue). C) Infrared spectra of the bosutinib isomer bound to Abl (black) and Src (red). D) The residues that comprise the ATP-binding site near the nitrile of bosutinib (black) are shown for our structure of Abl bound to bosutinib (yellow) and for that of Src bound to dasatinib (pdb code 3G5D, dark red).</p

Data collection and refinement statistics.

a<p>values in parentheses are for the highest resolution shell.</p

Structure of authentic bosutinib bound to the Abl tyrosine kinase domain.

<p>A) Interaction of bosutinib (blue) with the hinge region of Abl (yellow). For comparison, the binding modes of erlotinib (red) and gefitinib (pink) are also shown, and were obtained by aligning the structures of these compounds bound to EGFR (pdb codes 1M17 and 2ITY for erlotinib and gefitinib, respectively) on the hinge region of Abl. B) The interactions between bosutinib and T315 and V299 of Abl are shown. The residues T315 and V299 are shown as sticks and a yellow surface, and bosutinib is shown as blue sticks, with the 2F_o-F_c electron density map shown as a blue mesh. The T315I mutation is modeled as thin black sticks, and the resulting clash with bosutinib is shown as black dots. C) Binding curves for bosutinib binding to Src and the Src T338I mutant. The fluorescence intensity measured at 480 nm is plotted as a function of the total bosutinib concentration. The inset shows an expanded view of the binding curve for Src. The equilibrium dissociation constants were determined by a fitting procedure described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029828#s2" target="_blank">Materials and Methods</a>. D) Binding curves for vandetanib binding to Abl, Src and the Src T338I mutant. The fluorescence emission intensity measured at 440 nm, with excitation at 280 nm, is plotted as a function of the total vandetanib concentration. E) The conformation of the P-loop in our structure (shown in yellow, the two disordered residues are indicated as a dashed yellow line), compared to that observed in the imatinib cocrystal structure (pdb code 1IEP, shown in gray), and a substrate complex of Abl (pdb code 2G1T, shown in brown). The clash between Y253 and bosutinib that would result from the collapsed conformation of the P-loop is shown as black dots.</p

Experimental Quantification of Electrostatics in X–H···π Hydrogen Bonds

Hydrogen bonds are ubiquitous in chemistry and biology. The physical forces that govern hydrogen-bonding interactions have been heavily debated, with much of the discussion focused on the relative contributions of electrostatic vs quantum mechanical effects. In principle, the vibrational Stark effect, the response of a vibrational mode to electric field, can provide an experimental method for parsing such interactions into their electrostatic and nonelectrostatic components. In a previous study we showed that, in the case of relatively weak O–H···π hydrogen bonds, the O–H bond displays a linear response to an electric field, and we exploited this response to demonstrate that the interactions are dominated by electrostatics (Saggu, M.; Levinson, N. M.; Boxer, S. G. <i>J. Am. Chem. Soc.</i> <b>2011</b>, <i>133</i>, 17414–17419). Here we extend this work to other X–H···π interactions. We find that the response of the X–H vibrational probe to electric field appears to become increasingly nonlinear in the order O–H < N–H < S–H. The observed effects are consistent with differences in atomic polarizabilities of the X–H groups. Nonetheless, we find that the X–H stretching vibrations of the model compounds indole and thiophenol report quantitatively on the electric fields they experience when complexed with aromatic hydrogen-bond acceptors. These measurements can be used to estimate the electrostatic binding energies of the interactions, which are found to agree closely with the results of energy calculations. Taken together, these results highlight that with careful calibration vibrational probes can provide direct measurements of the electrostatic components of hydrogen bonds

The SH3 domain of Csk is required for dimerization.

<p>A) The constructs used in this paper. B) The results of size exclusion chromatography performed with the constructs shown in A. The elution volumes of molecular weight standards are indicated by black arrows.</p

The Csk SH3-SH3 dimer.

<p>A) A representative SH3-SH3 dimer from the crystal structure of full-length Csk (PDB code: 1K9A). Residues in the interface are highlighted. B) The results of size exclusion chromatography performed with constructs of Csk bearing mutations in the putative dimer interface.</p

Direct Measurements of Electric Fields in Weak OH···π Hydrogen Bonds

Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the π-cloud of benzene, and we used this as a model system to study the role of electric fields in weak OH···π hydrogen bonds. The effects of complex formation on the vibrational frequency of the phenol OH or OD stretches were measured in a series of benzene-based aromatic solvents. Large shifts are observed and these can be converted into electric fields via the measured vibrational Stark effect. A comparison of the measured fields with quantum chemical calculations demonstrates that calculations performed in the gas phase are surprisingly effective at capturing the electrostatics observed in solution. The results provide quantitative measurements of the magnitude of electric fields and electrostatic binding energies in these interactions and suggest that electrostatics dominate them. The combination of vibrational Stark effect (VSE) measurements of electric fields and high-level quantum chemistry calculations is a general strategy for quantifying and characterizing the origins of intermolecular interactions