title: Electrostatic ratchet in the protective antigen channel promotes anthrax toxin translocation.
abstract: Central to the power-stroke and brownian-ratchet mechanisms of protein translocation is the process through which nonequilibrium fluctuations are rectified or ratcheted by the molecular motor to transport substrate proteins along a specific axis. We investigated the ratchet mechanism using anthrax toxin as a model. Anthrax toxin is a tripartite toxin comprised of the protective antigen (PA) component, a homooligomeric transmembrane translocase, which translocates two other enzyme components, lethal factor (LF) and edema factor (EF), into the cytosol of the host cell under the proton motive force (PMF). The PA-binding domains of LF and EF (LF(N) and EF(N)) possess identical folds and similar solution stabilities; however, EF(N) translocates ∼10-200-fold slower than LF(N), depending on the electrical potential (Δψ) and chemical potential (ΔpH) compositions of the PMF. From an analysis of LF(N)/EF(N) chimera proteins, we identified two 10-residue cassettes comprised of charged sequence that were responsible for the impaired translocation kinetics of EF(N). These cassettes have nonspecific electrostatic requirements: one surprisingly prefers acidic residues when driven by either a Δψ or a ΔpH; the second requires basic residues only when driven by a Δψ. Through modeling and experiment, we identified a charged surface in the PA channel responsible for charge selectivity. The charged surface latches the substrate and promotes PMF-driven transport. We propose an electrostatic ratchet in the channel, comprised of opposing rings of charged residues, enforces directionality by interacting with charged cassettes in the substrate, thereby generating forces sufficient to drive unfolding.
authors.affiliation: California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, USA.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Wynia-Smith
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Brown
authors.forename: Gina
authors.initials: G
authors.lastname: Chirichella
authors.forename: Gigi
authors.initials: G
authors.lastname: Kemalyan
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Krantz
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008567:Membranes, Artificial; D008956:Models, Chemical; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018892:Proton-Motive Force; D055672:Static Electricity
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008567:Membranes, Artificial; C030325:anthrax toxin