Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model

송새미,김진식,이강석,하남출 한국미생물학회 2015 The journal of microbiology Vol.53 No.6

Gram-negative bacteria expel a wide range of toxic substances through tripartite drug efflux pumps consisting of an inner membrane transporter, an outer membrane channel protein, and a periplasmic adaptor protein. These pumps form tripartite assemblies which can span the entire cell envelope, including the inner and outer membranes. There have been controversial findings regarding the assembly of the individual components in tripartite drug efflux pumps. Recent structural and functional studies have advanced our understanding of the assembly and working mechanisms of the pumps. Here, we re-evaluate the assembly models based on recent structural and functional studies. In particular, this study focuses on the ‘adaptor bridging model’, highlighting the intermeshing cogwheel-like interactions between the tip regions of the outer membrane channel protein and the periplasmic adaptor protein in the hexameric assembly.

난배양성 미생물의 기능 분석 방법

김정명,송새미,전체옥,Kim, Jeong-Myeong,Song, Sae-Mi,Jeon, Che-Ok 한국미생물학회 2009 미생물학회지 Vol.45 No.1

미생물 군집 내의 미생물은 순수하게 배양된 미생물과는 다른 생리적 특징을 갖는다. 전통적으로 미생물 연구는 순수배양에 초점을 맞추어 이루어져 왔고 실제 생태계에 존재하는 대부분의 미생물들이 난배양성 미생물로 알려져 있다. 따라서 복잡한 미생물 군집에서 미생물의 기능에 대한 연구는 실질적으로 미진한 실정이다. 그러나 stable isotope probing (SIP), fluorescence in situ hybridization (FISH)와 microautoradiography (MAR)의 조합, isotope micrarray, 메타게노믹스 등의 새로운 분석방법들은 미생물 군집 내에서 난배양성 미생물의 기능 분석을 어느 정도 가능하게 해 주었다. 본 논문에서는 이들 방법 등에 대해 간단히 설명하고 좀 더 정확한 결과를 얻기 위한 최신 연구 동향을 소개하고자 한다. Microbes within complex communities show quite different physiology from pure cultured microbes. However, historically the study of microbes has focused on single species in pure culture and most of microbes are unculturable in our labs, so understanding of complex communities lags behind understanding of pure cultured cells. Methodologies including stable isotope probing (SIP), a combination of fluorescence in situ hybridization (FISH) and microautoradiography (MAR), isotope micrarray, and metagenomics have given insights into the uncultivated majority to link phylogenetic and functional information. Here, we review some of the most recent literatures, with an emphasis on methodological improvements to the sensitivity and utilities of these methods to link phylogeny and function in complex microbial communities.

Crystal Structure of the Regulatory Domain of AphB from Vibrio vulnificus, a Virulence Gene Regulator

박노라,송새미,최가람,장경구,조인성,최상호,하남출 한국분자세포생물학회 2017 Molecules and cells Vol.40 No.4

The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens Vibrio vulnificus and Vibrio cholerae. To date, the full-length AphB crystal structure of V. cholerae has been determined and characterized by a tetrameric assembly of AphB consisting of a DNA binding domain and a regulatory domain (RD). Although acidic pH and low oxygen tension might be in-volved in the activation of AphB, it remains unknown which ligand or stimulus activates AphB at the molecular level. In this study, we determine the crystal structure of the AphB RD from V. vulnificus under aerobic conditions without modification at the conserved cysteine residue of the RD, even in the presence of the oxidizing agent cumene hydroperoxide. A cysteine to serine amino acid residue mutant RD protein further confirmed that the cysteine residue is not involved in sensing oxidative stress in vitro. Interestingly, an unidentified small molecule was observed in the inter-subdomain cavity in the RD when the crystal was incubated with cumene hydroperoxide molecules, suggesting a new ligand-binding site. In addition, we confirmed the role of AphB in acid tolerance by observing an aphB-dependent in-crease in cadC transcript level when V. vulnificus was exposed to acidic pH. Our study contributes to the understanding of the AphB molecular mechanism in the process of recognizing the host environment.

Interaction between the α-Barrel Tip of Vibrio vulnificus TolC Homologs and AcrA Implies the Adapter Bridging Model

이승화,송새미,이민호,황순혜,김지선,하남출,이강석 한국미생물학회 2014 The journal of microbiology Vol.52 No.2

The AcrAB-TolC multidrug efflux pump confers resistanceto Escherichia coli against many antibiotics and toxic compounds. The TolC protein is an outer membrane factor thatparticipates in the formation of type I secretion systems. Thegenome of Vibrio vulnificus encodes two proteins homologousto the E. coli TolC, designated TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 partially complementthe E. coli TolC function and physically interactwith the membrane fusion protein AcrA, a component of theE. coli AcrAB-TolC efflux pump. Using site-directed mutationalanalyses and an in vivo cross-linking assay, we demonstratedthat the α-barrel tip region of TolC homologs playsa critical role in the formation of functional AcrAB-TolCefflux pumps. Our findings suggest the adapter bridgingmodel as a general assembly mechanism for tripartite drugefflux pumps in Gram-negative bacteria.

Crystal structure of Streptomyces coelicolor RraAS2, an unusual member of the RNase E inhibitor RraA protein family

박노라,허지훈,송새미,조인성,이강석,하남출 한국미생물학회 2017 The journal of microbiology Vol.55 No.5

Bacterial ribonuclease E (RNase E) plays a crucial role in theprocessing and decay of RNAs. A small protein named RraAnegatively regulates the activity of RNase E via protein-proteininteraction in various bacteria. Recently, RraAS1 and RraAS2,which are functional homologs of RraA from Escherichia coli,were identified in the Gram-positive species Streptomycescoelicolor. RraAS1 and RraAS2 inhibit RNase ES ribonucleaseactivity in S. coelicolor. RraAS1 and RraAS2 have a C-terminalextension region unlike typical bacterial RraA proteins. In this study, we present the crystal structure of RraAS2, exhibitinga hexamer arranged in a dimer of trimers, consistentwith size exclusion chromatographic results. Importantly,the C-terminal extension region formed a long α-helix at thejunction of the neighboring subunit, which is similar to thetrimeric RraA orthologs from Saccharomyces cerevisiae. Truncationof the C-terminal extension region resulted in loss ofRNase ES inhibition, demonstrating its crucial role. Our findingspresent the first bacterial RraA that has a hexamericassembly with a C-terminal extension α-helical region, whichplays an essential role in the regulation of RNase ES activityin S. coelicolor.

Crystal Structure of DsbA from Corynebacterium diphtheriae and Its Functional Implications for CueP in Gram-Positive Bacteria

엄시현,김진식,송새미,김남아,정성훈,하남출 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.8

In Gram-negative bacteria in the periplasmic space, the dimeric thioredoxin-fold protein DsbC isomerizes and reduces incorrect disulfide bonds of unfolded proteins, while the monomeric thioredoxin-fold protein DsbA introduces disulfide bonds in folding proteins. In the Gram-negative bacteria Salmonella enterica serovar Typhimurium, the reduced form of CueP scavenges the production of hydroxyl radicals in the copper-mediated Fenton reaction, and DsbC is responsible for keeping CueP in the reduced, active form. Some DsbA proteins fulfill the functions of DsbCs, which are not present in Gram-positive bacteria. In this study, we identified a DsbA homologous protein (CdDsbA) in the Corynebacterium diphtheriae genome and determined its crystal structure in the reduced condition at 1.5 Å resolution. CdDsbA consists of a monomeric thioredoxin-like fold with an inserted helical domain and unique N-terminal extended region. We confirmed that CdDsbA has disulfide bond isomerase/reductase activity, and we present evidence that the N-terminal extended region is not required for this activity and folding of the core DsbA-like domain. Furthermore, we found that CdDsbA could reduce CueP from C. diphtheriae.

The α-Barrel Tip Region of Escherichia coli TolC Homologs of Vibrio vulnificus Interacts with the MacA Protein to Form the Functional Macrolide-Specific Efflux Pump MacAB-TolC

이민호,김현리,송새미,주민주,이승화,김대영,한윤수,하남출,이강석 한국미생물학회 2013 The journal of microbiology Vol.51 No.2

TolC and its homologous family of proteins are outer membrane factors that are essential for exporting small molecules and toxins across the outer membrane in Gram-negative bacteria. Two open reading frames in the Vibrio vulnificus genome that encode proteins homologous to Escherichia coli TolC, designated TolCV1 and TolCV2, have 51.3% and 29.6%amino acid identity to TolC, respectively. In this study, we show that TolCV1 and TolCV2 functionally and physically interacted with the membrane fusion protein, MacA, a component of the macrolide-specific MacAB-TolC pump of E. coli. We further show that the conserved residues located at the aperture tip region of the α-hairpin of TolCV1 and TolCV2played an essential role in the formation of the functional MacAB-TolC pump using site-directed mutational analyses. Our findings suggest that these outer membrane factors have conserved tip-to-tip interaction with the MacA membrane fusion protein for action of the drug efflux pump in Gramnegative bacteria.

Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Suggests an Alternative Assembly Mode

김진식,하남출,정형섭,송새미,김혜연,이강석,현재경 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.2

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the -barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.

RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity

허지훈,김대영,주민주,이보은,서소진,이재진,송새미,염지현,하남출,이강석 한국미생물학회 2016 The journal of microbiology Vol.54 No.10

RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic‐producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.