Structural and Functional Analysis of the C-terminal DNA Binding Domain of the Salmonella typhimurium SPI-2 Response Regulator SsrB

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB (SsrB_C) by NMR and analyzed the effect of amino acid substitutions on function. We identified residues in the DNA recognition helix (Lys¹⁷⁹, Met¹⁸⁶) and the dimerization interface (Val¹⁹⁷, Leu²⁰¹) that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue in the N-terminal receiver domain was also identified (Cys⁴⁵), and the effect of Cys²⁰³ on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent activation of transcription. Disruption of the dimer interface by a C203E substitution reduces SsrB activity. Modification of Cys²⁰³ or Cys⁴⁵ may be an important mode of SsrB inactivation inside the host.Salmonella infections occur in a wide variety of vertebrate hosts and continue to be a major health problem worldwide for humans. Salmonella infection requires at least two pathogenicity islands, Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2),³ both of which encode type III secretion systems as well as secreted effectors, chaperones, and regulatory proteins (1–3). Genes located on SPI-1 are required for initial adherence to and invasion of intestinal epithelial cells (4). SPI-2 genes are required for intracellular survival, replication, and systemic infection of Salmonella (5, 6). SPI-2 consists of a 40-kb region located at 31 centisomes on the chromosome and contains ∼32 genes (7). SPI-2 genes are organized into functional clusters encoding regulatory, structural genes, effectors, and chaperones. To date 10 promoters of SPI-2 genes and SPI-2 co-regulated genes have been identified (8–10). These promoters, located upstream of the ssrA, ssrB, ssaB, sseA, ssaG, ssaM, sseI, srfN, sifA, and sifB genes, transcribe genes either individually or in large operons. Transcription from these promoters is activated by the SPI-2 encoded two-component signal transduction system, SsrA/SsrB (8, 11–13).Two-component signal transduction systems regulate gene expression in response to specific environmental signals (for review see Ref. 14). These systems represent the major paradigm for signal transduction in prokaryotes. They are frequently involved in regulating expression of virulence genes in pathogenic bacteria and are also present in the archaea, lower eukaryotes, and plants. In its simplest form, a two-component system contains a sensor kinase, often a membrane protein that functions in trans-membrane signaling, and a response regulator, usually a DNA-binding protein that regulates transcription. In Salmonella enterica serovar typhimurium, the SsrA/SsrB two-component system controls expression of SPI-2 genes as well as several non-SPI-2-encoded virulence genes (8, 11–13). These genes encode effector proteins that are secreted through the SPI-2 type III secretion system or are structural components of the secretory apparatus (5, 11).SsrA is a tripartite kinase composed of an ATP binding domain, a histidine phosphorylation domain, a receiver or phosphorylation domain also present in response regulators and a histidyl phosphotransfer domain. SsrA is located in the bacterial inner membrane and, based on homology to similar histidine kinases, has two transmembrane domains. These complex domain structures suggest a phosphorelay whereby the phosphoryl group is transferred via intramolecular reactions from the histidine to an aspartate, to a histidine, and then onto the conserved aspartate of the SsrB response regulator (8, 9, 13, 15).SsrB is a two-domain response regulator in the NarL/FixJ subfamily. The N-terminal receiver domain of SsrB contains the conserved aspartic acid phosphorylation site and the C-terminal effector domain binds DNA (13). The receiver domains are highly conserved, whereas the effector domains are more tailored to their specialized output functions. NarL, a response regulator involved in nitrogen sensing, is the first structure of this subfamily to be solved and it is also the first structure of a full-length response regulator (16). The structure elucidates many details of the activation mechanism, as the recognition helix is physically blocked by the N terminus. Phosphorylation drives a conformational change that relieves this inhibitory effect, exposing the recognition helix and promoting dimerization (17). SsrB functions by a similar mechanism (13). To activate transcription, SsrB must be phosphorylated at Asp⁵⁶ (13), yet surprisingly, overexpression can activate SPI-2 genes in the absence of the SsrA kinase (8, 13). This result indicates that alternative inputs in the form of additional kinases or small molecule phosphodonors such as acetyl phosphate must exist that can phosphorylate SsrB. In this manner the SsrA/SsrB system is uncoupled, as is expression of the ssrA/ssrB genes (9, 13).SsrB is homologous at the primary sequence level to several other two-component regulators for which structures have been determined, including NarL, RcsB, and DosR (16, 18–21). Although this subfamily shares significant structural similarity, important functional differences exist. DosR forms a unique tetramer on the DNA, forming a dimer of dimers, although its relevance in vivo remains to be established (21). NarL binds to its DNA targets as a homodimer, whereas RcsB can bind to DNA either as a homodimer or as a heterodimer with RcsA (22). NarL binds to a well conserved DNA consensus sequence (23), whereas the RcsB binding site is more degenerate (22), and it is difficult to discern a specific binding motif for SsrB_C (8). DosR binds to a pseudopalindrome that is GC-rich (19). The DNA binding domain of SsrB_C alone can function as a transcription factor in vivo (13), but the DNA binding domain of NarL cannot, even though it is capable of DNA binding (17). It was therefore of interest to determine how these differences in function might be reflected as structural differences between the subfamily members.In the present work, we solved the solution structure of SsrB_C by NMR and examined the effect of amino acid substitution on DNA binding, dimerization, and transcription. The C-terminal 75 amino acid residues of SsrB (SsrB_C) fold into a four-helix bundle, and the SsrB_C dimerization surface is similar to that of DosR, NarL, and RcsB. SsrB_C binds to regions of DNA that are AT-rich with poor sequence conservation. We identified residues in the DNA recognition helix and the dimerization interface that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue located in the N-terminal receiver domain was also identified, and the effect of Cys²⁰³ on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent transcriptional activation. Disruption of the dimer interface by substitution of Cys²⁰³ with a negatively charged residue substantially reduces SsrB activity. Cys²⁰³ modification may represent an important mode of SsrB inactivation inside the host.