Mechanistic and biophysical characterization of polymyxin resistance response regulator PmrA in Acinetobacter baumannii.

Introduction: Acinetobacter baumannii PmrAB is a crucial two-component regulatory system (TCS) that plays a vital role in conferring resistance to polymyxin. PmrA, a response regulator belonging to the OmpR/PhoB family, is composed of a C-terminal DNA-binding effector domain and an N-terminal receiver domain. The receiver domain can be phosphorylated by PmrB, a transmembrane sensor histidine kinase that interacts with PmrA. Once phosphorylated, PmrA undergoes a conformational change, resulting in the formation of a symmetric dimer in the receiver domain. This conformational change facilitates the recognition of promoter DNA by the DNA-binding domain of PmrA, leading to the activation of adaptive responses. Methods: X-ray crystallography was carried out to solve the structure of PmrA receiver domain. Electrophoretic mobility shift assay and Isothermal titration calorimetry were recruited to validate the interaction between the recombinant PmrA protein and target DNA. Field-emission scanning electron microscopy (FE-SEM) was employed to characterize the surface morphology of A. baumannii in both the PmrA knockout and mutation strains. Results: The receiver domain of PmrA follows the canonical α5ß5 response regulator assembly, which undergoes dimerization upon phosphorylation and activation. Beryllium trifluoride is utilized as an aspartate phosphorylation mimic in this process. Mutations involved in phosphorylation and dimerization significantly affected the expression of downstream pmrC and naxD genes. This impact resulted in an enhanced cell surface smoothness with fewer modifications, ultimately contributing to a decrease in colistin (polymyxin E) and polymyxin B resistance. Additionally, a conservative direct-repeat DNA PmrA binding sequence TTTAAGNNNNNTTTAAG was identified at the promoter region of the pmrC and naxD gene. These findings provide structural insights into the PmrA receiver domain and reveal the mechanism of polymyxin resistance, suggesting that PmrA could be a potential drug target to reverse polymyxin resistance in Acinetobacter baumannii.

The Functional Study of Response Regulator ArlR Mutants in Staphylococcus Aureus.

Staphylococcus aureus is a major cause of hospital-associated infections worldwide. The organism's ability to form biofilms has led to resistance against current treatment options such as beta-lactams, glycopeptides, and daptomycin. The ArlRS two-component system is a crucial regulatory system necessary for S. aureus autolysis, biofilm formation, capsule synthesis, and virulence. This study aims to investigate the role of the arlR deletion mutant in the detection and activation of S. aureus. We created an arlR deleted mutant and complementary strains and characterized their impact on the strains using partial growth measurement. The quantitative real-time PCR was performed to determine the expression of icaA, and the microscopic images of adherent cells were captured at the optical density of 600 to determine the primary bacterial adhesion. The biofilm formation assay was utilized to investigate the number of adherent cells using crystal violet staining. Eventually, the Triton X-100 autolysis assay was used to determine the influence of arlR on the cell autolytic activities. Our findings indicate that the deletion of arlR reduced the transcriptional expression of icaA but not icaR in the ica operon, leading to decrease in polysaccharide intercellular adhesin (PIA) synthesis. Compared to the wild-type and the complementary mutants, the arlR mutant exhibited decreased in biofilm production but increased autolysis. It concluded that the S. aureus response regulatory ArlR influences biofilm formation, agglutination, and autolysis. This work has significantly expanded our knowledge of the ArlRS two-component regulatory system and could aid in the development of novel antimicrobial strategies against S. aureus.

Structure and assembly of type VI secretion system cargo delivery vehicle.

Type VI secretion system is widely used in Gram-negative bacteria for injecting toxic effectors into neighboring prokaryotic or eukaryotic cells. Various effectors can be loaded onto the T6SS delivery tube via its core components: Hcp, VgrG, or PAAR. Here, we report 2.8-Å resolution cryo-EM structure of intact T6SS Hcp5-VgrG-PAAR cargo delivery system and crystal structure of unbound Hcp5 from B. fragilis NCTC 9343. Loading of Hcp5 hexameric ring onto VgrG causes expansion of its inner cavity and external surface, explaining how structural changes could be propagated to regulate co-polymerization and surrounding contractile sheath. High-affinity binding between Hcp and VgrG causes entropically unfavorable structuring of long loops. Furthermore, interactions between VgrG trimer and Hcp hexamer are asymmetric, with three of the six Hcp monomers exhibiting a major loop flip. Our study provides insights into the assembly, loading, and firing of T6SS nanomachine that contributes to bacterial inter-species competition and host interactions.

Mechanistic and structural insights into the bifunctional enzyme PaaY from Acinetobacter baumannii.

PaaY is a thioesterase that enables toxic metabolites to be degraded through the bacterial phenylacetic acid (PA) pathway. The Acinetobacter baumannii gene FQU82_01591 encodes PaaY, which we demonstrate to possess γ-carbonic anhydrase activity in addition to thioesterase activity. The crystal structure of AbPaaY in complex with bicarbonate reveals a homotrimer with a canonical γ-carbonic anhydrase active site. Thioesterase activity assays demonstrate a preference for lauroyl-CoA as a substrate. The AbPaaY trimer structure shows a unique domain-swapped C-termini, which increases the stability of the enzyme in vitro and decreases its susceptibility to proteolysis in vivo. The domain-swapped C-termini impact thioesterase substrate specificity and enzyme efficacy without affecting carbonic anhydrase activity. AbPaaY knockout reduced the growth of Acinetobacter in media containing PA, decreased biofilm formation, and impaired hydrogen peroxide resistance. Collectively, AbPaaY is a bifunctional enzyme that plays a key role in the metabolism, growth, and stress response mechanisms of A. baumannii.

Screening and epitope characterization of diagnostic nanobody against total and activated Bacteroides fragilis toxin.

Enterotoxigenic Bacteroides fragilis (ETBF) can rapidly secrete an enterotoxin termed B. fragilis toxin (BFT), which is thought to be the only recognized virulence factor in ETBF. ETBF can cause acute diarrhea, inflammatory bowel disease (IBD), colorectal cancer, and breast cancer. BFT is divided into three subtypes, BFT1, BFT2, and BFT3. BFT1 is the most widely distributed in human B. fragilis isolates. BFT can be used as a biomarker for predicting the inflammation-cancer transformation of intestine and breast. Nanobodies have the advantages of small structure, complete antigen recognition capacity, rapid selection via phage display technology, and can be massively produced in microbial expression systems. Nanobodies have become a powerful tool for medical diagnosis and treatment. This study focuses on screening and structural characterization of nanobodies targeting full length and active BFT. By constructing prokaryotic expression systems to obtain recombinant BFT1 protein, high purity BFT1 protein was used to immunize alpacas. Phage display technology was used to construct a phage display library. The positive clones were selected by bio-panning, and the isothermal titration calorimetry was used to select high-affinity nanobodies. Then the three-dimensional structures of BFT1:Nb2.82 and BFT1:Nb3.27 were solved by crystal X-ray diffraction. We got two kinds of nanobodies, Nb2.82 targeting the BFT1 prodomain and Nb3.27 recognizing the BFT1 catalytic domain. This study provides a new strategy for the early diagnosis of ETBF and the possibility for BFT as a biomarker for diagnosing diseases.

Progress in structural and functional study of the bacterial phenylacetic acid catabolic pathway, its role in pathogenicity and antibiotic resistance.

Phenylacetic acid (PAA) is a central intermediate metabolite involved in bacterial degradation of aromatic components. The bacterial PAA pathway mainly contains 12 enzymes and a transcriptional regulator, which are involved in biofilm formation and antimicrobial activity. They are present in approximately 16% of the sequenced bacterial genome. In this review, we have summarized the PAA distribution in microbes, recent structural and functional study progress of the enzyme families of the bacterial PAA pathway, and their role in bacterial pathogenicity and antibiotic resistance. The enzymes of the bacterial PAA pathway have shown potential as an antimicrobial drug target for biotechnological applications in metabolic engineering.

An Outer Membrane Protein YiaD Contributes to Adaptive Resistance of Meropenem in Acinetobacter baumannii.

Acinetobacter baumannii is an important nosocomial pathogen that can develop various resistance mechanisms to many antibiotics. However, little is known about how it evolves from an antibiotic sensitive to a resistant phenotype. In this study, we investigated the transition of outer membrane proteins (OMPs) under antibiotic stress and identified YiaD as an OMP marker involved in the development of adaptive resistance to meropenem (MEM) in A. baumannii. Following stimulation of a carbapenem-sensitive strain AB5116 with sub-MIC of MEM, yiaD showed significantly decreased expression, and this decrease continued with prolonged stimulation for 8 h. The downregulation of yiaD was not only observed in clinically sensitive strains but also in 45 carbapenem-resistant isolates that produced the ß-lactamases TEM and OXA-23. However, the extent of the reduction of yiaD expression in resistant strains was less than that in sensitive strains. Lack of yiaD resulted in a 4-fold increase in the MIC of AB5116 to MEM. The same level of depressed susceptibility induced by yiaD deletion was observed in both a growth curve test and a survival rate assay. Moreover, the colony shape became enlarged and irregular after loss of yiaD, and the biofilm formation ability of A. baumannii was influenced by YiaD. These results suggest that YiaD could respond to the stimulus of MEM in A. baumannii with a downregulation trend that kept pace with the prolonged stimulation time, indicating that it participates in various routes to benefit MEM resistance evolution in both carbapenem-sensitive and -resistant A. baumannii strains. IMPORTANCE Acinetobacter baumannii can develop various resistance mechanisms to carbapenems. However, the factors involved in the evolutionary process that leads from transition to the sensitive to resistant phenotype are not clear. The outer membrane protein YiaD of A. baumannii was downregulated under the stress of meropenem (MEM), and its expression level was continuously reduced with prolonged stimulation time. The downregulation of yiaD was not only observed in sensitive strains but also in carbapenem-resistant isolates producing the ß-lactamases TEM and OXA-23. However, the extent of yiaD reduction was less in resistant strains than in sensitive strains. Lack of yiaD resulted in an increased MEM MIC, enlarged and irregular colonies, and decreased biofilm formation ability. These results suggest that YiaD responds to MEM stimulus in A. baumannii and participates in the adaptive resistance of MEM in both carbapenem-sensitive and -resistant strains.

Non-canonical phosphorylation of Bmf by p38 MAPK promotes its apoptotic activity in anoikis.

Bmf contributes to the onset of anoikis by translocating from cytoskeleton to mitochondria when cells lose attachment to the extracellular matrix. However, the structural details of Bmf cytoskeleton tethering and the control of Bmf release upon loss of anchorage remained unknown. Here we showed that cell detachment induced rapid and sustained activation of p38 MAPK in mammary epithelial cell lines. Inhibition of p38 signaling or Bmf knockdown rescued anoikis. Activated p38 MAPK could directly phosphorylate Bmf at multiple sites including a non-proline-directed site threonine 72 (T72). Crystallographic studies revealed that Bmf T72 directly participated in DLC2 binding and its phosphorylation would block Bmf/DLC2 interaction through steric hindrance. Finally, we showed that phosphomimetic mutation of T72 enhanced Bmf apoptotic activity in vitro and in a knock-in mouse model. This work unraveled a novel regulatory mechanism of Bmf activity during anoikis and provided structural basis for Bmf cytoskeleton tethering and dissociation.

Proteolysis and multimerization regulate signaling along the two-component regulatory system AdeRS.

Bacterial two-component regulatory systems are ubiquitous environment-sensing signal transducers involved in pathogenesis and antibiotic resistance. The Acinetobacter baumannii two-component regulatory system AdeRS is made up of a sensor histidine kinase AdeS and a cognate response regulator AdeR, which together reduce repression of the multidrug-resistant efflux pump AdeABC. Herein we demonstrate that an N-terminal intrinsically disordered tail in AdeR is important for the upregulation of adeABC expression, although it greatly increases the susceptibility of AdeR to proteasome-mediated degradation. We also show that AdeS assembles into a hexameric state that is necessary for its full histidine kinase activity, which appears to occur via cis autophosphorylation. Taken together, this study demonstrates new structural mechanisms through which two-component systems can transduce environmental signals to impact gene expression and enlightens new potential antimicrobial approach by targeting two-component regulatory systems.

Development and Characterization of Nanobodies Targeting the Kupffer Cell.

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

DUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

Trimeric structure of the mouse Kupffer cell C-type lectin receptor Clec4f.

The C-type lectin receptor Clec4f has been identified as a specific surface marker for Kupffer cells, although its ortholog is absent in humans and its biological function remains elusive. Here, we report the crystal structure of a truncated mouse trimeric Clec4f. The orientation between the carbohydrate-recognition domain of Clec4f and its neck region differs from other C-type lectins, resulting in an observed distance of 45 Å between the glycan-binding sites within the Clec4f trimer. Interestingly, the trimeric coiled-coil interface within its heptad neck region contains multiple polyglutamine interactions instead of the predominantly hydrophobic leucine zipper found in other C-type lectin receptors. The Clec4f trimeric structure displays unique features regarding its assembly and ligand recognition, shedding light on the evolution and diversity of the C-type lectin family.

Deciphering the activation and recognition mechanisms of Staphylococcus aureus response regulator ArlR.

Staphylococcus aureus ArlRS is a key two-component regulatory system necessary for adhesion, biofilm formation, and virulence. The response regulator ArlR consists of a C-terminal DNA-binding effector domain and an N-terminal receiver domain that is phosphorylated by ArlS, the cognate transmembrane sensor histidine kinase. We demonstrate that the receiver domain of ArlR adopts the canonical α5ß5 response regulator assembly, which dimerizes upon activation, using beryllium trifluoride as an aspartate phosphorylation mimic. Activated ArlR recognizes a 20-bp imperfect inverted repeat sequence in the ica operon, which is involved in intercellular adhesion polysaccharide production. Crystal structures of the inactive and activated forms reveal that activation induces a significant conformational change in the ß4-α4 and ß5-α5-connecting loops, in which the α4 and α5 helices constitute the homodimerization interface. Crystal structures of the DNA-binding ArlR effector domain indicate that it is able to dimerize via a non-canonical ß1-ß2 hairpin domain swapping, raising the possibility of a new mechanism for signal transduction from the receiver domain to effector domain. Taken together, the current study provides structural insights into the activation of ArlR and its recognition, adding to the diversity of response regulation mechanisms that may inspire novel antimicrobial strategies specifically targeting Staphylococcus.

NIRF-Molecular Imaging with Synovial Macrophages-Targeting Vsig4 Nanobody for Disease Monitoring in a Mouse Model of Arthritis.

Nanobody against V-set and Ig domain-containing 4 (Vsig4) on tissue macrophages, such as synovial macrophages, could visualize joint inflammation in multiple experimental arthritis models via single-photon emission computed tomography imaging. Here, we further addressed the specificity and assessed the potential for arthritis monitoring using near-infrared fluorescence (NIRF) Cy7-labeled Vsig4 nanobody (Cy7-Nb119). In vivo NIRF-imaging of collagen-induced arthritis (CIA) was performed using Cy7-Nb119. Signals obtained with Cy7-Nb119 or isotope control Cy7-NbBCII10 were compared in joints of naive mice versus CIA mice. In addition, pathological microscopy and fluorescence microscopy were used to validate the arthritis development in CIA. Cy7-Nb119 accumulated in inflamed joints of CIA mice, but not the naive mice. Development of symptoms in CIA was reflected in increased joint accumulation of Cy7-Nb119, which correlated with the conventional measurements of disease. Vsig4 is co-expressed with F4/80, indicating targeting of the increasing number of synovial macrophages associated with the severity of inflammation by the Vsig4 nanobody. NIRF imaging with Cy7-Nb119 allows specific assessment of inflammation in experimental arthritis and provides complementary information to clinical scoring for quantitative, non-invasive and economical monitoring of the pathological process. Nanobody labelled with fluorescence can also be used for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

A tetrameric protein scaffold as a nano-carrier of antitumor peptides for cancer therapy.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, PMIBcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein PMIBcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM. PMIBcr/Abl-R6 effectively induced apoptosis of HCT116 p53+/+ cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our protein-based delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy and is likely applicable to the development of many other peptide therapeutics to target a great variety of intracellular protein-protein interactions responsible for disease initiation and progression.

Publisher Correction: ERK-mediated phosphorylation regulates SOX10 sumoylation and targets expression in mutant BRAF melanoma.

In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following: The National Basic Research Program (2015CB553602 to J.L.), the National Natural Science Foundation of China (31570777, 91649106, 31770917 to J.L.) and Tianjin Applied Basic and Frontier Tech Major Project (12JCZDJC34400 to J.L.) and Tianjin Higher Education Sci-Tech Development Project (20112D05 to J.L.).

Development and optimization of a tumor targeting system based on microbial synthesized PHA biopolymers and PhaP mediated functional modification.

Polyhydroxyalkanoate (PHA) is a class of microbial synthesized biodegradable and biocompatible aliphatic polymer which has been developed into nanoparticles (NPs) for sustained release of hydrophobic compounds. Taking advantage of the natural PHA binding protein PhaP which could be steadily adsorbed onto PHA NPs through hydrophobic interaction, a tumor targeting system was developed in this study by presenting an epidermal growth factor receptor (EGFR)-targeting peptide (ETP) on the surface of PHA NPs, via PhaP mediated adsorption. To reveal the effects of residual emulsifiers on PhaP mediated ETP modification and optimize the tumor targeting capacity of the system, a novel emulsifier-free PHA NPs (EF-NPs) was fabricated together with other two kinds of conventional emulsifier-required PHA NPs (PVA-NPs and P68-NPs, which were prepared with poly(vinyl alcohol) (PVA) and Pluronic F68 as emulsifiers, respectively). By analyzing the surface hydrophobicity, the amount of adsorbed fusion protein, and the cellular uptake of all kinds of PHA NPs, our results demonstrated that EF-NPs with stronger surface hydrophobicity were the most proper formulation for further PhaP mediated ETP functionalization. The residual PVA and Pluronic F68 affected the modification efficiency and secondary structure of ETP-PhaP fusion protein, and finally obstructed the targeting effect of ETP-PhaP modified PVA-NPs and P68-NPs to EGFR over-expressed tumor cells. The animal experiment further confirmed the effectiveness and feasibility of in vivo application of ETP-PhaP functionalized EF-NPs, indicating that it could be served as a promising tumor targeting system with satisfactory EGFR targeting ability. This PhaP mediated bio-modification process also opens a wide way for developing various PHA-based targeting systems by presenting different tumor or other tissue-specific targeting peptides.

ERK-mediated phosphorylation regulates SOX10 sumoylation and targets expression in mutant BRAF melanoma.

In human mutant BRAF melanoma cells, the stemness transcription factor FOXD3 is rapidly induced by inhibition of ERK1/2 signaling and mediates adaptive resistance to RAF inhibitors. However, the mechanism underlying ERK signaling control of FOXD3 expression remains unknown. Here we show that SOX10 is both necessary and sufficient for RAF inhibitor-induced expression of FOXD3 in mutant BRAF melanoma cells. SOX10 activates the transcription of FOXD3 by binding to a regulatory element in FOXD3 promoter. Phosphorylation of SOX10 by ERK inhibits its transcription activity toward multiple target genes by interfering with the sumoylation of SOX10 at K55, which is essential for its transcription activity. Finally, depletion of SOX10 sensitizes mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo. Thus, our work discovers a novel phosphorylation-dependent regulatory mechanism of SOX10 transcription activity and completes an ERK1/2/SOX10/FOXD3/ERBB3 axis that mediates adaptive resistance to RAF inhibitors in mutant BRAF melanoma cells.

Mechanistic insight into how multidrug resistant Acinetobacter baumannii response regulator AdeR recognizes an intercistronic region.

AdeR-AdeS is a two-component regulatory system, which controls expression of the adeABC efflux pump involved in Acinetobacter baumannii multidrug resistance. AdeR is a response regulator consisting of an N-terminal receiver domain and a C-terminal DNA-binding-domain. AdeR binds to a direct-repeat DNA in the intercistronic region between adeR and adeABC. We demonstrate a markedly high affinity binding between unphosphorylated AdeR and DNA with a dissociation constant of 20 nM. In addition, we provide a 2.75 Å crystal structure of AdeR DNA-binding-domain complexed with the intercistronic DNA. This structure shows that the α3 and ß hairpin formed by ß5-ß6 interacts with the major and minor groove of the DNA, which in turn leads to the introduction of a bend. The AdeR receiver domain structure revealed a dimerization motif mediated by a gearwheel-like structure involving the D108F109-R122 motif through cation π stack interaction. The structure of AdeR receiver domain bound with magnesium indicated a conserved Glu19Asp20-Asp63 magnesium-binding motif, and revealed that the potential phosphorylation site Asp63OD1 forms a hydrogen bond with Lys112. We thus dissected the mechanism of how AdeR recognizes the intercistronic DNA, which leads to a diverse mode of response regulation. Unlocking the AdeRS mechanism provides ways to circumvent A. baumannii antibiotic resistance.

Crystal structure of master biofilm regulator CsgD regulatory domain reveals an atypical receiver domain.

The master regulator CsgD switches planktonic growth to biofilm formation by activating synthesis of curli fimbriae and cellulose in Enterobacteriaceae. CsgD was classified to be the LuxR response regulatory family, while its cognate sensor histidine kinase has not been identified yet. CsgD consists of a C-terminal DNA binding domain and an N-terminal regulatory domain that provokes the upstream signal transduction to further modulate its function. We provide the crystal structure of Salmonella Typhimurium CsgD regulatory domain, which reveals an atypical ß5α5 response regulatory receiver domain folding with the α2 helix representing as a disorder loop compared to the LuxR/FixJ canonical response regulator, and the structure indicated a noteworthy α5 helix similar to the non-canonical master regulator VpsT receiver domain α6. CsgD regulatory domain assembles with two dimerization interfaces mainly through α1 and α5, which has shown similarity to the c-di-GMP independent and stabilized dimerization interface of VpsT from Vibrio cholerae respectively. The potential phosphorylation site D59 is directly involved in the interaction of interfaces I and mutagenesis studies indicated that both dimerization interfaces could be crucial for CsgD activity. The structure reveals important molecular details for the dimerization assembly of CsgD and will shed new insight into its regulation mechanism.