Characterization of the ABC methionine transporter from Neisseria meningitidis reveals that lipidated MetQ is required for interaction.

NmMetQ is a substrate-binding protein (SBP) from Neisseria meningitidis that has been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are localized to the periplasmic space to promote interaction with their cognate ABC transporter embedded in the bacterial inner membrane. To elucidate the roles of NmMetQ, we characterized NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the structures of NmMetNI in the presence and absence of lipo-NmMetQ. Based on our data, we propose that NmMetQ tethers to membranes via a lipid anchor and has dual function and localization, playing a role in NmMetNI-mediated transport at the inner membrane and moonlighting on the bacterial surface.

Structures of the Neisseria meningitides methionine-binding protein MetQ in substrate-free form and bound to l- and d-methionine isomers.

The bacterial periplasmic methionine-binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate-free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l- and d-methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate-free form and in complexes with l-methionine and with d-methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate-free (N238A) and substrate-bound N. meningitides MetQ are related by a "Venus-fly trap" hinge-type movement of the two domains accompanying methionine binding and dissociation. l- and d-methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand-free MetQ associates with the ATP-bound form of MetNI ∼40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.

Noncanonical role for the binding protein in substrate uptake by the MetNI methionine ATP Binding Cassette (ABC) transporter.

The Escherichia coli methionine ABC transporter MetNI exhibits both high-affinity transport toward l-methionine and broad specificity toward methionine derivatives, including d-methionine. In this work, we characterize the transport of d-methionine derivatives by the MetNI transporter. Unexpectedly, the N229A substrate-binding deficient variant of the cognate binding protein MetQ was found to support high MetNI transport activity toward d-selenomethionine. We determined the crystal structure at 2.95 Šresolution of the ATPγS-bound MetNIQ complex in the outward-facing conformation with the N229A apo MetQ variant. This structure revealed conformational changes in MetQ providing substrate access through the binding protein to the transmembrane translocation pathway. MetQ likely mediates uptake of methionine derivatives through two mechanisms: in the methionine-bound form delivering substrate from the periplasm to the transporter (the canonical mechanism) and in the apo form by facilitating ligand binding when complexed to the transporter (the noncanonical mechanism). This dual role for substrate-binding proteins is proposed to provide a kinetic strategy for ABC transporters to transport both high- and low-affinity substrates present in a physiological concentration range.

The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex.

Despite the ubiquitous role of ATP-binding cassette (ABC) importers in nutrient uptake, only the Escherichia coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250-253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nm. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Šresolution), the dissociation constant for complex formation with MetNI is measured to be ∼40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system.

Open and shut: crystal structures of the dodecylmaltoside solubilized mechanosensitive channel of small conductance from Escherichia coli and Helicobacter pylori at 4.4 Å and 4.1 Å resolutions.

The mechanosensitive channel of small conductance (MscS) contributes to the survival of bacteria during osmotic downshock by transiently opening large diameter pores for the efflux of cellular contents before the membrane ruptures. Two crystal structures of the Escherichia coli MscS are currently available, the wild type protein in a nonconducting state at 3.7 Å resolution (Bass et al., Science 2002; 298:1582-1587) and the Ala106Val variant in an open state at 3.45 Å resolution (Wang et al., Science 2008; 321:1179-1183). Both structures used protein solubilized in the detergent fos-choline-14. We report here crystal structures of MscS from E. coli and Helicobacter pylori solubilized in the detergent ß-dodecylmaltoside at resolutions of 4.4 and 4.2 Å, respectively. While the cytoplasmic domains are unchanged in these structures, distinct conformations of the transmembrane domains are observed. Intriguingly, ß-dodecylmaltoside solubilized wild type E. coli MscS adopts the open state structure of A106V E. coli MscS, while H. pylori MscS resembles the nonconducting state structure observed for fos-choline-14 solubilized E. coli MscS. These results highlight the sensitivity of membrane protein conformational equilibria to variations in detergent, crystallization conditions, and protein sequence.