Manuscript submitted to eLife Characterization of the ABC 1 methionine transporter from 2 Neisseria meningitidis reveals that 3 MetQ is a lipoprotein 4 Naima G. Sharaf 1,2,* , Mona Shahgholi 1 , Esther Kim 1 , Jeffrey Y. Lai 1,2 , David 5 VanderVelde 1 , Allen T. Lee 1,2 , Douglas C. Rees 1,2,* 6 *For correspondence: dcrees@caltech.edu (DCR); ngsharaf@caltech.edu (NGS) 1 California Institute of Technology, Division of Chemistry and Chemical Engineering 7 114-96, California Institute of Technology, Pasadena, CA 91125 USA; 2 Howard Hughes 8 Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA 9 10 Abstract NmMetQ is a substrate binding protein (SBP) from Neisseria meningitidis that has 11 been identified as a surface-exposed candidate antigen for meningococcal vaccines. However, 12 this location for NmMetQ challenges the prevailing view that SBPs in Gram-negative bacteria are 13 localized to the periplasmic space to promote interaction with their cognate ABC transporter 14 embedded in the bacterial inner membrane. To address the roles of NmMetQ, we characterized 15 NmMetQ with and without its cognate ABC transporter (NmMetNI). Here, we show that NmMetQ 16 is a lipoprotein (lipo-NmMetQ) that binds multiple methionine analogs and stimulates the ATPase 17 activity of NmMetNI. Using single-particle electron cryo-microscopy, we determined the 18 structures of NmMetNI in the absence and presence of lipo-NmMetQ. Based on our data, we 19 propose that NmMetQ tethers to membranes via a lipid anchor and has dual function/topology, 20 playing a role in NmMetNI-mediated transport at the inner-membrane in addition to 21 moonlighting functions on the bacterial surface. 22 23 Introduction 24 The substrate binding protein NmMetQ from the human pathogen Neisseria meningitidis has been 25 identified as a surface-exposed candidate antigen for the meningococcal vaccine (Pizza et al., 2000). 26 Subsequently, NmMetQ was shown to interact with human brain microvascular endothelial cells 27 (Kánová et al., 2018), potentially acting as an adhesin. However, the surface-topology of NmMetQ 28 challenges the prevailing view that substrate binding proteins (SBPs) reside in the periplasm of 29 Gram-negative bacteria (Thomas and Tampé, 2020), binding and delivering molecules to cognate 30 ATP-Binding Cassette (ABC) transporters in the inner-membrane (IM). Several questions arise from 31 these studies: Has NmMetQ lost its ABC transporter-dependent function in the IM? and How does 32 NmMetQ become embedded in the outer membrane (OM) surface of the bacterium? 33 The ABC transporter-dependent role of SBPs has been well characterized for multiple ABC trans- 34 porter systems (Hollenstein et al., 2007; Oldham et al., 2013; Sabrialabed et al., 2020; Liu et al., 35 2020; Nguyen et al., 2018; de Boer et al., 2019). These studies reveal conserved SBP-dependent 36 characteristics, including that the SBP is largely responsible for substrate delivery to the ABC trans- 37 porter, with concomitant stimulation of the transport coupled ATPase activity. Structural stud- 38 ies have shown that SBPs dock to the periplasmic surface of the transporter’s transmembrane 39 1 of 20 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted May 4, 2021. ; https://doi.org/10.1101/2021.05.04.442564 doi: bioRxiv preprint