Eric Kool, Postdoctoral Faculty Sponsor
Diverse Reagent Scaffolds Provide Differential Selectivity of 2'-OH Acylation in RNA.
Journal of the American Chemical Society
RNA 2'-OH acylation is widely used both for mapping structure and for conjugating RNA, generally relying on selective reactions with unpaired nucleotides over paired ones. Common reagents for this acylation have been chiefly restricted to two similar aryl scaffolds, leaving open the question of how more broadly varied reagent structure might affect selectivity. Here, we prepared a set of 10 structurally diverse acylimidazole reagents and employed deep sequencing to profile their reactivity and selectivity in an RNA library of systematically varied structure. We show that structure-directed reactivity profiles vary significantly with the reagent scaffold, and we document new acylating agents that have altered selectivity profiles, including reagents that show elevated selectivity within loops, as well as compounds with reduced off-target reactivity in loop closing base pairs. Interestingly, we also show that the simplest reagent (acetylimidazole) is cell permeable and is small enough to map RNA structure in the presence of protein contacts that block other reagents. Finally, we describe reagents that show elevated selectivity within small loops, with applications in site-selective labeling. The results provide new tools for improved conjugation and mapping of RNA.
View details for DOI 10.1021/jacs.2c09040
View details for PubMedID 36542611
Acylation probing of "generic" RNA libraries reveals critical influence of loop constraints on reactivity.
Cell chemical biology
The reactivity of RNA 2'-OH acylation is broadly useful both in probing structure and in preparing conjugates. To date, this reactivity has been analyzed in limited sets of biological RNA sequences, leaving open questions of how reactivity varies inherently without regard to sequence in structured contexts. We constructed and probed "generic" structured RNA libraries using homogeneous loop sequences, employing deep sequencing to carry out a systematic survey of reactivity. We find a wide range of RNA reactivities among single-stranded sequences, with nearest neighbors playing substantial roles. Remarkably, certain small loops are found to be far more reactive on average (up to 4,000-fold) than single-stranded RNAs, due to conformational constraints that enhance reactivity. Among loops, we observe large variations in reactivity based on size, type, and position. The results lend insights into RNA designs for achieving high-efficiency local conjugation and provide new opportunities to refine structure analysis.
View details for DOI 10.1016/j.chembiol.2022.05.005
View details for PubMedID 35662395
The Right Tool for the Job: A Chemical and Genetic Toolkit for Interrogating DCLK1 Function
CELL CHEMICAL BIOLOGY
2020; 27 (10): 1221–23
Cell permeable, small molecule inhibitors are powerful tools for interrogating kinase function and validating drug targets. In this issue of Cell Chemical Biology, Liu and colleagues (2020) describe the development of a toolkit containing a highly selective DCLK1 inhibitor and complementary DCLK1 mutants for interrogating DCLK1-dependent cellular processes.
View details for DOI 10.1016/j.chembiol.2020.09.007
View details for Web of Science ID 000581602800001
View details for PubMedID 33065025
Parallel Chemoselective Profiling for Mapping Protein Structure
CELL CHEMICAL BIOLOGY
2020; 27 (8): 1084-+
Solution-based structural techniques complement high-resolution structural data by providing insight into the oft-missed links between protein structure and dynamics. Here, we present Parallel Chemoselective Profiling, a solution-based structural method for characterizing protein structure and dynamics. Our method utilizes deep mutational scanning saturation mutagenesis data to install amino acid residues with specific chemistries at defined positions on the solvent-exposed surface of a protein. Differences in the extent of labeling of installed mutant residues are quantified using targeted mass spectrometry, reporting on each residue's local environment and structural dynamics. Using our method, we studied how conformation-selective, ATP-competitive inhibitors affect the local and global structure and dynamics of full-length Src kinase. Our results highlight how parallel chemoselective profiling can be used to study a dynamic multi-domain protein, and suggest that our method will be a useful addition to the relatively small toolkit of existing protein footprinting techniques.
View details for DOI 10.1016/j.chembiol.2020.06.014
View details for Web of Science ID 000561681400015
View details for PubMedID 32649906
View details for PubMedCentralID PMC7484201
How ATP-Competitive Inhibitors Allosterically Modulate Tyrosine Kinases That Contain a Src-like Regulatory Architecture
ACS CHEMICAL BIOLOGY
2020; 15 (7): 2005–16
Small molecule kinase inhibitors that stabilize distinct ATP binding site conformations can differentially modulate the global conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects generalize to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src's regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix αC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src's helix αC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Our study highlights that an ATP-competitive inhibitor's interactions with helix αC can have a major influence on the global conformation of some tyrosine kinases.
View details for DOI 10.1021/acschembio.0c00429
View details for Web of Science ID 000551550000033
View details for PubMedID 32479050
View details for PubMedCentralID PMC7700871
Chemoproteomic Method for Profiling Inhibitor-Bound Kinase Complexes
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2019; 141 (30): 11912–22
Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein's functions affects the state of the cell is not well-understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. Here, we describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans-cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src's cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src's interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src's localization and interactions in situ. Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets.
View details for DOI 10.1021/jacs.9b02963
View details for Web of Science ID 000479018200031
View details for PubMedID 31274292
View details for PubMedCentralID PMC6688853
Targeting Dynamic ATP-Binding Site Features Allows Discrimination between Highly Homologous Protein Kinases
ACS CHEMICAL BIOLOGY
2019; 14 (6): 1249–59
ATP-competitive inhibitors that demonstrate exquisite selectivity for specific members of the human kinome have been developed. Despite this success, the identification of highly selective inhibitors is still very challenging, and it is often not possible to rationally engineer selectivity between the ATP-binding sites of kinases, especially among closely related family members. Src-family kinases (SFKs) are a highly homologous family of eight multidomain, nonreceptor tyrosine kinases that play general and specialized roles in numerous cellular processes. The high sequence and functional similarities between SFK members make it hard to rationalize how selectivity can be gained with inhibitors that target the ATP-binding site. Here, we describe the development of a series of inhibitors that are highly selective for the ATP-binding sites of the SFKs Lyn and Hck over other SFKs. By biochemically characterizing how these selective ATP-competitive inhibitors allosterically influence the global conformation of SFKs, we demonstrate that they most likely interact with a binding pocket created by the movement of the conformationally flexible helix αC in the ATP-binding site. With a series of sequence swap experiments, we show that sensitivity to this class of selective inhibitors is due to the identity of residues that control the conformational flexibility of helix αC rather than any specific ATP-binding site interactions. Thus, the ATP-binding sites of highly homologous kinases can be discriminated by targeting heterogeneity within conformationally flexible regions.
View details for DOI 10.1021/acschembio.9b00214
View details for Web of Science ID 000473115900023
View details for PubMedID 31038916
View details for PubMedCentralID PMC6642640
A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions
2019; 74 (2): 393-+
Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.
View details for DOI 10.1016/j.molcel.2019.02.003
View details for Web of Science ID 000465184200016
View details for PubMedID 30956043
View details for PubMedCentralID PMC6474823
Catalytic asymmetric synthesis of the Colorado potato beetle pheromone and its enantiomer
2014; 25 (8): 591–95
View details for DOI 10.1016/j.tetasy.2013.12.018
View details for Web of Science ID 000336708800001