ABSTRACT
Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.
Subject(s)
Recombinant Fusion Proteins/metabolism , Small Molecule Libraries/metabolism , Binding Sites , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drosophila Proteins/genetics , HEK293 Cells , Humans , Models, Molecular , Mutation , Protein Stability , Temperature , UbiquitinationABSTRACT
NanoRNase (Nrn) specifically degrades nucleoside 3',5'-bisphosphate and the very short RNA, nanoRNA, during the final step of mRNA degradation. The crystal structure of Nrn in complex with a reaction product GMP was determined. The overall structure consists of two domains that are interconnected by a flexible loop and form a cleft. Two Mn²âº ions are coordinated by conserved residues in the DHH motif of the N-terminal domain. GMP binds near the DHHA1 motif region in the C-terminal domain. Our structure enables us to predict the substrate-bound form of Nrn as well as other DHH/DHHA1 phosphoesterase family proteins.
Subject(s)
Bacterial Proteins/chemistry , Bacteroides/enzymology , Ribonucleases/chemistry , Amino Acid Motifs , Binding Sites , Crystallography, X-Ray , Escherichia coli/metabolism , Ligands , Manganese/chemistry , Models, Molecular , Protein Binding , Protein Conformation , RNA/chemistry , RNA, Messenger/metabolism , Substrate SpecificityABSTRACT
The Morita-Baylis-Hillman reaction forms a carbon-carbon bond between the α-carbon of a conjugated carbonyl compound and a carbon electrophile. The reaction mechanism involves Michael addition of a nucleophile catalyst at the carbonyl ß-carbon, followed by bond formation with the electrophile and catalyst disassociation to release the product. We used Rosetta to design 48 proteins containing active sites predicted to carry out this mechanism, of which two show catalytic activity by mass spectrometry (MS). Substrate labeling measured by MS and site-directed mutagenesis experiments show that the designed active-site residues are responsible for activity, although rate acceleration over background is modest. To characterize the designed proteins, we developed a fluorescence-based screen for intermediate formation in cell lysates, carried out microsecond molecular dynamics simulations, and solved X-ray crystal structures. These data indicate a partially formed active site and suggest several clear avenues for designing more active catalysts.
