Cooperation of two distinct coupling proteins creates chemosensory network connections Samar Abedrabboa, Juan Castellona, Kieran D. Collinsa, Kevin S. Johnsona, and Karen M. Ottemanna,1 aDepartment of Microbiology and Environmental Toxicology, University of California, Santa Cruz, CA 95064 Edited by Caroline S. Harwood, University of Washington, Seattle, WA, and approved January 25, 2017 (received for review November 2, 2016) Although it is appreciated that bacterial chemotaxis systems rely arrays and positive cooperativity (3, 8). Thus, coupling proteins on coupling, also called scaffold, proteins to both connect input participate in two types of CheA interactions that are vital for receptors with output kinases and build interkinase connections chemotaxis function. that allow signal amplification, it is not yet clear why many systems Although these types of CheW–CheA interactions have been use more than one coupling protein. We examined the distinct elucidated in the single coupling protein E. coli system, it is not functions for multiple coupling proteins in the bacterial chemotaxis yet known how and whether these interactions differ in multi- system of Helicobacter pylori, which requires two nonredundant cou- coupling protein systems. To date, the best-studied multi- coupling protein system is that of Bacillus subtilis, a microbe that pling proteins for chemotaxis: CheW and CheV1, a hybrid of a CheW – and a phosphorylatable receiver domain. We report that CheV1 and uses one CheV and one CheW to both perform receptor kinase CheW have largely redundant abilities to interact with chemorecep- coupling in a somewhat functionally redundant manner (13, 14). There is evidence suggesting that some chemoreceptors might tors and the CheA kinase, and both similarly activated CheA’skinase operate with particular coupling proteins, displaying greater af- activity. We discovered, however, that they are not redundant for finity for one coupling protein than for another (15, 16). CheW formation of the higher order chemoreceptor arrays that are known – and CheV may also have some affinity bias toward different to form via CheA CheW interactions. In support of this possibility, we chemoreceptors (17). found that CheW and CheV1 interact with each other and with CheA The human pathogen Helicobacter pylori has a chemotaxis independent of the chemoreceptors. Therefore, it seems that some system with four chemoreceptors called Tlps (TlpA, TlpB, TlpC, microbes have modified array formation to require CheW and CheV1. and TlpD), a CheA kinase, a CheY response regulator, and— Our data suggest that multiple coupling proteins may be used to relevant to this work—multiple coupling proteins (18, 19). Two MICROBIOLOGY provide flexibility in the chemoreceptor array formation. of the H. pylori coupling proteins, CheW and CheV1, are critical for wild-type chemotaxis, acting in a nonredundant manner. signal transduction | scaffold | chemotaxis | chemoreceptor arrays Mutants lacking cheW or cheV1 appear unable to activate the CheA kinase as they swim without changing direction and are oupling or scaffold proteins provide critical connections be- either completely (cheW) or severely (cheV1) compromised in a Ctween input receptors and output kinases in many types of soft agar chemotaxis assay (9, 20). H. pylori also possesses two signal transduction pathways (1–3). These connections confer other CheV-type coupling proteins, but these play only minor multiple advantages such as cooperativity, signaling complex as- roles in chemotaxis (9, 20). Because H. pylori CheW and CheV1 were both essential for chemotaxis in a nonredundant manner, sembly, and protein localization (2). Indeed, many cellular signaling we thought it ideal to dissect how these contribute to chemotaxis. systems use multiple coupling proteins to fine tune these advan- We initiated our work analyzing the protein interaction net- tages, a process that has been well studied in eukaryotic systems (2, work of CheW and CheV1 as well as their ability to activate and 4, 5). Bacterial chemotaxis is an example of a prokaryotic system control CheA’s kinase function. We found that they had nearly that relies on coupling proteins of the CheW family to connect identical abilities in these regards, which did not explain why chemoreceptors to the CheA kinase and build connections that both were needed. However, when we examined their roles in create multiprotein chemoreceptor arrays. Many bacterial chemo- assembly of the polar chemosensory array, we found that both taxis systems possess multiple coupling proteins (6, 7), but the functions and advantages of having more than one coupling protein Significance for these systems are not well understood. The core bacterial chemotaxis sensory unit is composed of a chemoreceptor, a CheW family coupling protein, and the CheA Signal transduction systems are important pathways that or- output kinase (6). The coupling protein allows the chemore- ganisms use to sense and respond to their environments. ceptors to control the CheA kinase and promotes connections Chemotaxis is controlled by a signal transduction system that between core units (3, 6–8). Chemotaxis coupling proteins have allows bacteria to coordinate their movement in response to two basic architectures, CheW or CheV. CheW is a single do- their environment. This response requires proper assembly and main protein with two defined subdomains, and CheV proteins localization of large multiprotein chemotaxis complexes, which are hybrids that add a C-terminal response regulator-like domain are built by interactions between coupling proteins. The sig- (Rec) to an N-terminal CheW domain (3, 9). nificance of having multiple types of coupling proteins in a Chemotaxis core sensory units exist in cells as large multi- single signal transduction system is poorly understood. Here, protein arrays at the poles (8, 10–12). Connections that form we show that multiple coupling proteins allow bacteria to these arrays have been elucidated in the single-coupling protein build superprotein interaction networks and localize them to system of Escherichia coli and are driven by interactions between cell poles, a role that is required for optimal chemotaxis. CheW and a subdomain of CheA called P5, which is a structural mimic of CheW. These connections are vital for array formation, Author contributions: S.A., J.C., and K.M.O. designed research; S.A., J.C., and K.S.J. per- kinase control, and positive cooperativity that allow small signals formed research; K.D.C. contributed new reagents/analytic tools; S.A. and K.M.O. ana- to be greatly amplified (11). There are two documented types of lyzed data; and S.A. and K.M.O. wrote the paper. CheW–CheA P5 interactions. Interactions at interface 1 occur The authors declare no conflict of interest. between CheA P5 subdomain 1 and CheW subdomain 2 and lead This article is a PNAS Direct Submission. to control of CheA kinase activity (3, 8). Interactions at interface 1To whom correspondence should be addressed. Email: [email protected]. 2 occur between CheA P5 subdomain 2 and CheW subdomain 1 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and lead to intercomplex connections that build chemoreceptor 1073/pnas.1618227114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1618227114 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 were required and fulfilled nonredundant roles in these inter- CheW were able to interact with one another and with them- actions. Our data demonstrate that some microbes use multiple selves. Lastly, CheV1 interacted with CheV2 and CheV3. coupling proteins to build the polar chemoreceptor supercluster The BACTH protein interactions were verified using coim- and thus suggest this aspect of chemotaxis may be fine-tunable by munoprecipitation with purified proteins. Consistent with the modulating the levels or activities of these coupling proteins. BACTH, both CheW and CheV1 interacted with CheA and H. pylori’s cytoplasmic chemoreceptor, TlpD (Fig. 2 A and B). It Results is interesting to note that CheW did interact with the TlpD CheW and CheV1 Both Form Direct Interactions with CheA and chemoreceptor in the coimmunoprecipitation experiments but not in the BACTH experiments. Based on these data, it seems Chemoreceptors. To gain an understanding of why H. pylori re- ’ quires two coupling proteins, we first characterized the protein– possible that the BACTH cloning could have affected CheW s protein interaction network of H. pylori CheV1 and CheW. We structure and thus its ability to interact with TlpD. All H. pylori identified directly interacting proteins using the bacterial ade- chemoreceptors have highly similar signaling regions that con- nylate cyclase two-hybrid (BACTH) system (21). For this ap- serve residues that interact with coupling proteins in other sys- tems (Fig. S1). Because of the high degree of similarity and the proach, CheV1 and CheW were fused to the N or C terminus of fact that TlpD is soluble in its full-length form, we used TlpD to T25 fragments, and CheV1, CheW, CheA, CheV2, CheV3, and characterize chemoreceptor–coupling protein interactions. It is the chemoreceptors (TlpA, TlpB, and TlpD) were fused to the possible, however, that CheW and CheV1 interact with the other bait T18 fragments. We found that both CheV1 and CheW chemoreceptors with varying strength or preference (7, 17). Fi- displayed interactions with themselves, the other coupling pro- nally, we saw that both CheV1 and CheW interacted