proteins STRUCTURE O FUNCTION O BIOINFORMATICS Identification of a cation transport pathway in Neisseria meningitidis PorB Christof Kattner, 1y Jan Zaucha, 2y Frank Jaenecke, 1 Ulrich Zachariae, 2 * and Mikio Tanabe 1 * 1 HALOmem, Institut fu ¨r Biochemie und Biotechnologie, Martin-Luther-Universita ¨t Halle-Wittenberg, Halle (Saale), Germany 2 SUPA, School of Physics and Astronomy, The University of Edinburgh, Edinburgh, United Kingdom INTRODUCTION Neisseria meningitidis and Neisseria gonorrhoeae are the only species from the genus Neisseria that cause serious human diseases, namely, meningitis and gonorrhea. Through interaction with blood plasma and exudate fluid, they are capable of invading epithelial and endo- thelial cells. 1 The Neisserial porin PorB is one of the most abundant proteins in their outer membrane. To- gether with PorA, both proteins represent up to 60% of the total outer membrane protein content in these two pathogenic species. 2,3 Porins such as PorB form aqueous diffusion channels through the outer membrane. They are responsible for the essential exchange of polar solute molecules between the bacteria and their surrounding environment. 2 Addi- tionally, PorB has a unique pathogenic function, since it can translocate from the bacterial outer membrane into host cell membranes. 4–7 Although this process remains largely enigmatic, PorB may share features of the integra- tion pathway with the voltage-dependent anion channel (VDAC), 4,5 and it has been suggested that it can further integrate into the inner mitochondrial membrane. 7 This may ultimately have an impact on host cell apoptosis. 7,8 After insertion into host membranes, the activity of PorB is regulated by noncovalent binding of ATP and GTP. 9 Nucleotide binding stabilizes the channel in a mostly open state, with increased flux relative to the closed state that is usually induced by the transmembrane potential present in these membranes. 7,9 PorB also acts as a pathogen-associated molecular pattern (PAMP), activating the immune response Additional Supporting Information may be found in the online version of this article. y These authors have contributed equally to this work. Grant sponsor: Bundesministerium fu ¨r Bildung und Forschung (BMBF) ZIK pro- gram; Grant number: FKZ 03Z2HN21; Grant sponsors: Scottish Universities Physics Alliance and the UK National Physical Laboratory. *Correspondence to: Ulrich Zachariae; SUPA, School of Physics and Astronomy, The King’s Buildings, Mayfield Road, Edinburgh EH9 3JZ, United Kingdom. E-mail: uzachari@ph.ed.ac.uk or Mikio Tanabe; HALOmem, Membrane Protein Biochemistry, Institut fu ¨r Biochemie und Biotechnologie, Martin-Luther-Universita ¨t Halle-Wittenberg, Kurt-Mothes-Str.3 D-06120 Halle (Saale), Germany. E-mail: mt@halomem.de Received 18 October 2012; Revised 6 December 2012; Accepted 8 December 2012 Published online 19 December 2012 in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1002/prot.24241 ABSTRACT Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute transloca- tion pathways through this channel. Although PorB has been electrophysiologically characterized as anion-selective, cation translocation through nucleotide-bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 A ˚ resolu- tion. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs 1 , which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs 1 form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mito- chondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmem- brane potential gradient and thereby induce apoptosis. Proteins 2013; 81:830–840. V V C 2012 Wiley Periodicals, Inc. Key words: outer membrane protein; Neisseria meningitidis; ion transport; crystal structure; molecular dynamics. 830 PROTEINS V V C 2012 WILEY PERIODICALS, INC.