Quantitative analysis of ERK2 interactions with substrate proteins: roles for kinase docking domains and activity in determining binding affinity.

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) proteins regulate a variety of cellular functions, including cell proliferation and differentiation, by interacting with and phosphorylating substrate proteins. Two docking sites, common docking (CD/ED) domain and F-site recruitment site (FRS), on ERK proteins have been identified. Specific interactions with the CD/ED domain and the FRS occur with substrates containing a docking site for ERK and JNK, LXL (DEJL) motif (D-domain) and a docking site for ERK, FXF (DEF) motif (F-site), respectively. However, the relative contributions of the ERK docking sites in mediating substrate interactions that allow efficient phosphate transfer are largely unknown. In these studies, we provide a quantitative analysis of ERK2 interactions with substrates using surface plasmon resonance to measure real time protein-protein interactions. ERK2 interacted with ELK-1 (DEF and DEJL motifs), RSK-1 (DEJL motif), and c-Fos (DEF motif) with K(D) values of 0.25, 0.15, and 0.97 µM, respectively. CD/ED domain mutations inhibited interactions with ELK-1 and RSK-1 by 6-fold but had no effect on interactions with c-Fos. Select mutations in FRS residues differentially inhibited ELK-1 or c-Fos interactions with ERK2 but had little effect on RSK-1 interactions. Mutations in both the ED and FRS docking sites completely inhibited ELK-1 interactions but had no effect on interactions with stathmin, an ERK substrate whose docking site is unknown. The phosphorylation status of ERK2 did not affect interactions with RSK-1 or c-Fos but did inhibit interactions with ELK-1 and stathmin. These studies provide a quantitative evaluation of specific docking domains involved in mediating interactions between ERK2 and protein substrates and define the contributions of these interactions to phosphate transfer.

Functional characterization of the Shigella dysenteriae heme ABC transporter.

The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.

Characterization of the outer membrane receptor ShuA from the heme uptake system of Shigella dysenteriae. Substrate specificity and identification of the heme protein ligands.

Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.