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Crystal Structures of the Burkholderia Multivorans Hopanoid Transporter Hpnn

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Crystal Structures of the Burkholderia Multivorans Hopanoid Transporter Hpnn Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN Nitin Kumara,1, Chih-Chia Sub,1, Tsung-Han Choub, Abhijith Radhakrishnana, Jared A. Delmarb, Kanagalaghatta R. Rajashankarc,d, and Edward W. Yua,b,2 aDepartment of Chemistry, Iowa State University, Ames, IA 50011; bDepartment of Physics and Astronomy, Iowa State University, IA 50011; cNortheastern Collaborative Access Team, Argonne National Laboratory, Argonne, IL 60439; and dDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850 Edited by Eric Gouaux, Oregon Health and Science University, Portland, OR, and approved May 15, 2017 (received for review November 30, 2016) Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative critical line of defense against antimicrobial agents in Burkholderia opportunisitic bacteria that are capable of causing serious diseases, species is the permeability barrier of the outer membrane. Most of mainly in immunocompromised individuals. Bcc pathogens are in- these species contain a modified lipopolysaccharide, which results in trinsically resistant to multiple antibiotics, including β-lactams, ami- polymyxin resistance (13). In addition, the permeability of the major noglycosides, fluoroquinolones, and polymyxins. They are major outer membrane porin channel Omp38 appears to be low for an- pathogens in patients with cystic fibrosis (CF) and can cause severe tibiotics (14). The presence of a large number of multidrug efflux necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis pumps, belonging to the resistance-nodulation-cell division (RND) is one of the major mechanisms involved in multiple antimicrobial superfamily, also plays a major role in the intrinsic resistance to a resistance of Bcc pathogens. The hpnN gene of B. multivorans en- variety of antimicrobials. It has been found that some Bcc bacteria codes an integral membrane protein of the HpnN family of trans- harbor between 11 and 16 hydrophobic/amphiphile efflux 1 porters, which is responsible for shuttling hopanoids to the outer (HAE1)-RND pumps that are responsible for drug efflux (13). B. multivorans membrane. Here, we report crystal structures of In B. multivorans, it has been reported that hopanoids play a HpnN, revealing a dimeric molecule with an overall butterfly shape. predominant role in supporting membrane stability and barrier Each subunit of the transporter contains 12 transmembrane helices function, thus participating in multidrug resistance (9, 11). A and two periplasmic loops that suggest a plausible pathway for mutant strain of B. multivorans lacking the ability to synthesize substrate transport. Further analyses indicate that HpnN is capable hopanoids exhibits hypersensitivity to polymyxin B and colistin (9). of shuttling hopanoid virulence factors from the outer leaflet of the Hopanoids are pentacyclic triterpenoid lipids that are sterol ana- inner membrane to the periplasm. Taken together, our data suggest logs in prokaryotic membranes (15–17). Like cholesterols in that the HpnN transporter is critical for multidrug resistance and cell eukaryotic membranes, hopanoids are capable of inserting in wall remodeling in Burkholderia. bacterial membranes and contributing to their stability and stiff- ness (18). Hopanoids help membranes withstand damaging stress hopanoid transport | HpnN transporter | Burkholderia multivorans | multidrug resistance | cell wall remodeling conditions, including high temperature, low pH, and the presence of antibiotics (9, 11, 12, 18). Not all bacteria produce hopanoids, Burkholderia multivorans but they play a vital role in those that do make them. It has been he successful human pathogen is a shown that hopanoid production plays an important role in the member of the Burkholderia cepacia complex (Bcc) that T physiology and pathogenesis of B. cenocepacia (12, 19). causes pneumonia in immunocompromised individuals with un- derlying lung diseases, such as cystic fibrosis (CF) and chronic granulomatous disease (CGD) (1, 2). Bcc consists of a group of Significance at least 17 closely related Gram-negative bacteria with extreme genetic capacity and metabolic diversity. All Bcc members can Bcc bacteria are intrinsically resistant to multiple antibiotics. They trigger chronic airway infections in patients with CF and have are major pathogens in patients with cystic fibrosis (CF) and can emerged as opportunistic pulmonary pathogens (3). Burkholderia cause severe necrotizing pneumonia, which is often fatal. Hopa- cenocepacia and B. multivorans are the two most commonly noid biosynthesis is one of the major mechanisms involved in hpnN isolated species (4, 5), which are threats for outbreaks. Bcc in- multiple antimicrobial resistance of Bcc pathogens. The gene B. multivorans fections in patients with CF are associated with enhanced mor- of encodes an integral membrane protein of the bidity and mortality. They also have the capacity to cause rapid HpnN family of transporters, which is responsible for shuttling clinical deterioration with septicemia that leads to death. Several hopanoids to the outer membrane. Here, we report crystal struc- tures of B. multivorans HpnN that indicate a plausible pathway for outbreaks of B. multivorans causing severe morbidity and mor- hopanoid transport. Overall our data suggest a novel mechanism tality in both patients with CF and patients without CF have for hopanoid transport involved in cell wall remodeling, which is occurred (6–8). In 2013, the rapid emergence of a ceftazidime- critical for mediating multidrug resistance in Burkholderia. resistant strain of B. multivorans in a patient with CF was iden- tified in the United States (7). It was found that the resistant Author contributions: C.-C.S. and E.W.Y. designed research; N.K., C.-C.S., and E.W.Y. per- strain maintained dominance, resulting in an overall decline in formed research; N.K., C.-C.S., T.-H.C., A.R., J.A.D., K.R.R., and E.W.Y. analyzed data; and patient health and treatment efficiency. Subsequently, a wide- C.-C.S. and E.W.Y. wrote the paper. BIOCHEMISTRY spread outbreak of infection caused by B. multivorans was The authors declare no conflict of interest. reported from the Czech Republic (8). Surprisingly, this out- This article is a PNAS Direct Submission. break of B. multivorans affected patients without CF with 24% Freely available online through the PNAS open access option. B. multivorans mortality rate, indicating that is a significant and Data deposition: The atomic coordinates and structure factors have been deposited in the emerging threat beyond patients with CF. Protein Data Bank, www.pdb.org [PDB ID code 5KHN (form I) and 5KHS (form II)]. Bcc pathogens are intrinsically resistant to a broad range of 1N.K. and C.-C.S. contributed equally to this work. antimicrobials, including β-lactams, fluoroquinolones, amino- 2To whom correspondence should be addressed. Email: [email protected]. glycosides, polymyxins, and cationic peptides, creating a major This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. challenge to the treatment of Bcc pulmonary infections (9–12). A 1073/pnas.1619660114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619660114 PNAS | June 20, 2017 | vol. 114 | no. 25 | 6557–6562 Downloaded by guest on October 1, 2021 PD3 Despite the importance of hopanoids in bacteria, the mecha- α16 α7 nism of intracellular hopanoid trafficking for cell wall remodel- A B PD4 α17 ing has not been explored. A subfamily of the RND superfamily α6 α5 of transporters (20), termed hopanoid biosynthesis-associated α19 α18 α10 α8 PD2 RND (HpnN) transporters (21), are responsible for shuttling α15 α4 α20 β2 α14 hopanoids from the cytoplasmic membrane to the outer mem- β1 β3 β7 β6 brane of Gram-negative bacteria (22). Typically, an RND efflux α9 α3 α13 β4 β5 PD1 pump works in conjunction with a periplasmic membrane fusion α2 α21 protein (MFP) (23–25). However, the HpnN-subfamily trans- α11 α1 α12TM8 porters do not seem to associate with any of these MFPs. As an TM12 TM7 initial step to elucidate the mechanism of hopanoid transport, we TM2 here present the crystal structure of the B. multivorans HpnN TM1 transporter that is essential for cell wall biogenesis in this path- TM3 TM11 TM TM ogen. A combination of the 3D structure and genetic analysis TM6 TM5 TM4 allows us to identify important residues for the function of this TM10 membrane protein. TM9 Results Overall Structure of B. multivorans HpnN. B. multivorans HpnN C PD4 α16 α6 PD3 consists of 877 amino acids (SI Appendix,Fig.S1). Two distinct α17α18 α8 α7 conformations of HpnN (forms I and II) were captured in two α19 different forms of crystals (SI Appendix,TableS1). In each struc- ture, two monomers were found in the asymmetric unit arranged as α15 α5 a dimer (SI Appendix, Figs. S2 and S3). The dimeric form of HpnN α10 α20 β1 β5 in the crystal lattice is in good agreement with the oligomerization β2 β6 α4 α14 α9 α21 PD1 β4 β3 PD2 state of this membrane protein in detergent solution, in which it β7 SI Appendix assemblesasadimer( ,Fig.S4). Overall, the topology α3 of HpnN is unique. The HpnN dimer is butterfly shaped with a Periplasm α2 α13 α11 twofold symmetry axis perpendicular to the membrane plane (Fig. α1 α12 1A). The overall structure of HpnN indicates that this membrane protein mainly constitutes the transmembrane and periplasmic Inner membrane TM11 TM12 TM10 TM1b TM2 TM4 TM5 TM7b TM8 TM9 domains. Viewed in parallel to the membrane, the dimer is about TM3 TM6 110 Å tall, 100 Å wide, and 52 Å thick. Each protomer of HpnN in the dimer contains 12 trans- N TM1a TM7a C membrane helices (TMs 1–12 and TMs 1′–12′, respectively). In Cytoplasm addition, the monomer possesses a large periplasmic domain formed by two periplasmic loops between TMs 1 and 2 (loop 1), Fig. 1. Structure of the B. mulitvorans HpnN transporter. (A) Ribbon dia- and between TMs 7 and 8 (loop 2). Loop 1 is composed of 11 gram of a dimer of HpnN viewed in the membrane plane.
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