Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.

No abstract

No abstract

Targeting eukaryotic proteins for deamidation modification is increasingly appreciated as a general bacterial virulence mechanism. Here, we present an atomic view of how a bacterial deamidase effector, cycle-inhibiting factor homolog in Burkholderia pseudomallei (CHBP), recognizes its host targets, ubiquitin (Ub) and Ub-like neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), and catalyzes site-specific deamidation. Crystal structures of CHBP-Ub/NEDD8 complexes show that Ub and NEDD8 are similarly cradled by a large cleft in CHBP with four contacting surfaces. The pattern of Ub/NEDD8 recognition by CHBP resembles that by the E1 activation enzyme, which critically involves the Lys-11 surface in Ub/NEDD8. Close examination of the papain-like catalytic center reveals structural determinants of CHBP being an obligate glutamine deamidase. Molecular-dynamics simulation identifies Gln-31/ Glu-31 of Ub/NEDD8 as one key determinant of CHBP substrate preference for NEDD8. Inspired by the idea of using the unique bacterial activity as a tool, we further discover that CHBP-catalyzed NEDD8 deamidation triggers macrophage-specific apoptosis, which predicts a previously unknown macrophage-specific proapoptotic signal that is negatively regulated by neddylation-mediated protein ubiquitination/degradation.Cullin neddylation | type III secretion system | enteropathogenic E. coli | transglutamination | MLN4924 P rotein deamidation refers to hydrolysis of the side-chain carboxamide of a glutamine or rarely an asparagine. The related transamidation involves sequential deamidation and incorporation of a lysine ε-amine, resulting in cross-linking of two polypeptides (1). Humans have eight structurally related transglutaminases (TG1-7 and factor XIII) that regulate many biological processes. TGs can catalyze deamidation in the absence of a proper amine. Other promiscuous or dedicated enzymes capable of performing deamidation are also present in humans (2).Deamidation is more widely used in microorganisms. In bacterial chemosensory system, CheB from Escherichia coli/Salmonella enterica (3) and CheD from most nonenteric chemotactic bacteria (4) catalyze site-specific deamidation of chemotaxis receptors. Many secreted bacterial toxins also have the deamidase activity. Cytotoxic necrotizing factor 1/2 (CNF1/2) from certain virulent E. coli strains deamidate Gln-63/61 in Rho GTPases, rendering constitutively active GTPases and altered actin cytoskeleton (5, 6). Pasteurella multocida toxin (PMT) activates heterotrimeric G proteins by deamidating a conserved Gln in Gα (7). Cycle-inhibiting factor (Cif) from enteropathogenic E. coli (EPEC) and Cif homolog in Burkholderia pseudomallei (CHBP), both delivered into host cells through the type III secretion system (TTSS), induce a cytopathic effect of cell cycle arrest and actin stress fiber formation on epithelial cells. The Cif/CHBP family deamidates a conserved Gln-40 in host ubiquitin (Ub) and Ub-like protein (UBL) neural precursor cell expressed, developmentally downregu...