Conformational Coupling between Receptor and Kinase Binding Sites through a Conserved Salt Bridge in a Signaling Complex Scaffold Protein

et al. (2013) Conformational Coupling between Receptor and Kinase Binding Sites through a Conserved Salt Bridge in a Signaling Complex Scaffold Protein. PLoS Comput Biol 9(11): e1003337. doi:10.1371/journal.pcbi.1003337 Conformational Coupling between Receptor and Kinase Binding Sites through a Conserved Salt Bridge in a Signaling Complex Scaffold Protein Davi R. Ortega 0 Guoya Mo 0 Kwangwoon Lee 0 Hongjun Zhou 0 Jerome Baudry 0 Frederick W. Dahlquist 0 Igor B. Zhulin 0 Christopher V. Rao, University of Illinois at Urbana-Champaign, United States of America 0 1 Joint Institute for Computational Sciences, University of Tennessee - Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America, 2 Department of Physics, University of Tennessee, Knoxville, Tennessee, United States of America, 3 Department of Chemistry and Biochemistry, University of California, Santa Barbara, California, United States of America, 4 Department of Biochemistry and Cell and Molecular Biology, University of Tennessee, Knoxville, Tennessee, United States of America , 5 Center for Molecular Biophysics , University of Tennessee - Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America, 6 Department of Microbiology, University of Tennessee , Knoxville, Tennessee , United States of America Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD), NMR spectroscopy, and circular dichroism (CD), we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold. - Funding: This work was supported by the National Institutes of Health grants GM07225 (to IBZ) and GM059544 (to FWD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The Escherichia coli chemotaxis pathway employs dedicated chemoreceptors that are anchored in the membrane and detect signals from both outside and inside the cell [1]. Chemoreceptors relay this information to the CheA histidine kinase, which then communicates the information to its cognate response regulator CheY. In a phosphorylated form, the CheY protein binds to flagellar motors to cause a change in the direction of its rotation, thus converting the initial signal detected by chemoreceptors into a behavioral response a change in the swimming direction. This pathway also employs the receptor-modifying enzymes CheB and CheR as well as the CheZ phosphatase, which acts on CheY [2]. The key features of this remarkable system include high sensitivity, wide dynamic range, signal integration, memory, and precise adaptation [37], all of which are consequences of a highly ordered arrangement of chemoreceptor and kinase proteins at the cell pole [4,8,9]. The geometry of a hexagonal array with a lattice spacing of 12 nm is conserved over long evolutionary distances [9], indicating the importance of precise interactions among members of the complex. In addition to the chemoreceptors and the CheA kinase, this complex also contains the CheW protein, which is interchangeably referred to as a docking, scaffold, coupling, or adaptor protein [1012]. The crystal structure of CheW [10,13,14] reveals a fold composed of two five-stranded b-barrel subdomains connected by a hydrophobic core. Within the chemotaxis signaling complex, the CheW fold is present not only as a stand-alone adaptor but also as a homologous domain within the CheA kinase [15]. Furthermore, the two subdomains of CheW are topologically similar to the SH3 domain [15], which is widely distributed among scaffold proteins in eukaryotic signal transduction systems [16]. Thus, elucidating the structure/function relationships of CheW will have a broader impact in understanding the role of scaffold proteins in signal transduction system in all organisms. CheW is required for proper activation of the kinase by the chemoreceptor [17] and is essential for the formation of the chemotaxis complex [18]. Overexpression of CheW disrupts formation of chemoreceptor trimers by blocking trimer contacts [12,19,20], thereby impairing chemotaxis [21]. The binding sites for CheA and the chemoreceptor on CheW have been mapped using various experimental approaches [12,19,2224]. The overall results were consistent with CheW being a scaffold protein. However, the replacement of Arg62 (throughout the text, numbers Signal transduction is a universal biological process and a common target of drug design. The chemotaxis machinery in Escherichia coli is a model signal transduction system, and the CheW protein is one of its core components. CheW is thought to work as a scaffold protein that mediates the formation of the signaling complex with the CheA histidine kinase and the chemoreceptors. A mutation targeting a highly conserved residue, Arg62, impairs chemotaxis while maintaining normal binding affinity for both partners, suggesting that CheW might play a more complex role than previously proposed. Using a series of molecular dynamics simulations, we found that the residue Arg62 can form a stable salt bridge with another highly conserved residue, Glu38. We determined that this bridge does not contribute to the overall stability of the protein. However, the bridge stabilizes the local backbone structure of CheW and stabilizes the relative position of the binding sites for the chemoreceptor and kinase. The geometry of these interactions appears to be vital for the function of the signaling complex. We validated and complemented our computational findings using NMR spectroscopy and circular dichroism analysis. are for E. coli CheW) with His, which moderately affected in vitro binding affinity of CheW for both its binding partners, completely abolishes chemotaxis. This finding indicates that CheW plays a role in addition to holding CheA and the (...truncated)