Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN

Abstract

Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.

Crystal Structures of Membrane Transporter MmpL3, an Anti-TB Drug Target

“…Two putative pathways for substrate entry into MmpL3 are shown in Figure S2D. It is suggested that substrates located in the inner leaflet of the membrane can be transported across the membrane through the transmembrane groove at the periphery of transmembrane domain (Kumar et al, 2017;Li et al, 2016b;Oswald et al, 2016), wherein MmpL3 acts as flippase (Xu et al, 2017). Alternatively, TMM located in the outer leaflet of the membrane could gain access to G F that opens into the periplasm, which is gated by hydrophilic residues to recognize the trehalose head of TMM.…”

“…MmpL3 with a fused T4 lysozyme is monomeric in the crystalline state. However, this contrasts with AcrB (Murakami et al, 2002), a classic RND superfamily member involved in bacterial multidrug resistance, and CmpL1 (a homolog from Corynebacterium glutamicum) (Belardinelli et al, 2016), both of which have been reported as trimers, while another RND family member, NPC1, is a monomer (Gong et al, 2016) and HpnN is a dimer (Kumar et al, 2017). To determine the quaternary structure of MmpL3 in the absence of T4 lysozyme, full-length MmpL3 was expressed and purified ( Figure S1D).…”

Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched

“…Among bacterial RND homologs of known structure, the overall domain organization of PTCH1 most resembles that of the Burkholderia multivorans transporter HpnN, required for hopanoid export to the outer membrane (Kumar et al, 2017). Both PTCH1 and HpnN contain one alphabeta sandwich fold in each of their extracellular/periplasmic domains, with an interrupting sequence inserted between helix 1 and strand 2 ( Fig.…”

“…S6B). In addition, HpnN presents as a dimer during purification and crystallization (Kumar et al, 2017), thus displaying the same oligomeric state as PTCH1 (albeit with a distinct dimer architecture). Dimeric structure thus may not be a particularly rare feature of extended RND family members, even though the most extensively studied member of the family, AcrB, functions as a trimer.…”

“…5B). In HpnN, a structurally homologous groove on the side of TM1 has been proposed as the substrate entry site, and is continuous with the cavity in one conformation (Kumar et al, 2017). The residues surrounding these three densities seen in PTCH1 structure are primarily hydrophobic ( Fig.…”