Mechanistic characterization for c-jun-N-Terminal Kinase 1alpha1.

c-jun-N-terminal kinase 1alpha1 (JNK1alpha1) is a serine/threonine kinase of the mitogen-activated protein (MAP) kinase family that phosphorylates protein transcription factors after activation by a variety of environmental stressors. In this study, the kinetic mechanism for JNK1alpha1 phosphorylation of activating transcription factor 2 (ATF2) was determined utilizing steady-state kinetics in the presence and absence of both ATF2 and ATP competitive inhibitors. Data from initial velocity studies were consistent with a sequential mechanism for JNK1alpha1.

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII).

Kinetic mechanism and inhibitor characterization for c-jun-N-terminal kinase 3alpha1.

c-jun-N-Terminal kinase 3alpha1 (JNK3alpha1) is a mitogen-activated protein (MAP) kinase family member expressed primarily in the brain that phosphorylates protein transcription factors including c-jun and activating transcription factor 2 (ATF2) upon activation by a variety of stress-based stimuli. In this study, the kinetic mechanism for JNK3alpha1 was determined via initial velocity patterns in the presence and absence of both ATP and ATF2 competitive inhibitors. Peptide inhibitors from both ATF2 (peptide 1) and JNK-interacting protein 1 (JIP-1) (peptide 3), derived from the homologous delta-domain JNK docking sequence, inhibited JNK3alpha1 activity in a competitive fashion versus ATF2 while being pure noncompetitive toward ATP.

Synthesis of potent bicyclic bisarylimidazole c-Jun N-terminal kinase inhibitors by catalytic C-H bond activation.

The efficient preparation of the privileged bicyclic bis-arylimidazole kinase inhibitor scaffold was accomplished using rhodium-catalyzed C-H activation and intramolecular alkylation. The key C-H activation/alkylation step represents one of the first evaluations of acyclic stereocontrol in catalyzed C-H activation/olefin alkylation processes. Several inhibitors of JNK3 were prepared using this sequence, with the most potent inhibitor having an IC value of 1.

Selective recognition of synthetic lysine and meso-diaminopimelic acid-type peptidoglycan fragments by human peptidoglycan recognition proteins I{alpha} and S.

The interactions of a range of synthetic peptidoglycan derivatives with PGRP-Ialpha and PGRP-S have been studied in real-time using surface plasmon resonance. A dissociation constant of K(D) = 62 mum was obtained for the interaction of peptidoglycan recognition protein (PGRP)-Ialpha with the lysine-containing muramyl pentapeptide (compound 6). The normalized data for the lysine-containing muramyl tetra- (compound 5) and pentapeptide (compound 6) showed that these compounds have similar affinities, whereas a much lower affinity for muramyl tripeptide (compound 3) was measured.

Crystal structure of a peptidoglycan recognition protein (PGRP) in complex with a muramyl tripeptide from Gram-positive bacteria.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyse, bacterial peptidoglycans (PGNs). We determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the conserved core of lysine-type PGNs. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end could accommodate N-acetylglucosamine.

Structural basis for peptidoglycan binding by peptidoglycan recognition proteins.

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern-recognition receptors of the innate immune system that bind and, in some cases, hydrolyze bacterial PGNs. We determined the crystal structure, at 2.30-A resolution, of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the core of lysine-type PGNs from Gram-positive bacteria.

Multivalency and the mode of action of bacterial sialidases.

Although complex modular proteins are encountered frequently in a variety of biological systems, their occurrence in biocatalysis has not been widely appreciated. Here, we describe that bacterial sialidases, which have both a catalytic and carbohydrate-binding domain, can hydrolyze polyvalent substrates with much greater catalytic efficiency than their monovalent counterparts. The enhancement of catalytic efficiency was due to a much smaller Michaelis constant and rationalized by a model in which the catalytic and lectin domains interact simultaneously with the polyvalent substrate, leading to an enhancement of affinity.