A Receptor-based Switch that Regulates Anthrax Toxin Pore Formation

Citation: Pilpa RM, Bayrhuber M, Marlett JM, Riek R, Young JAT ( A Receptor-based Switch that Regulates Anthrax Toxin Pore Formation Rosemarie M. Pilpa 0 Monika Bayrhuber 0 John M. Marlett 0 Roland Riek 0 John A. 0 Steven R. Blanke, University of Illinois, United States of America 0 1 Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, California, United States of America, 2 Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, United States of America, 3 Laboratory of Physical Chemistry, ETH Z u rich , Zu rich , Switzerland Cellular receptors can act as molecular switches, regulating the sensitivity of microbial proteins to conformational changes that promote cellular entry. The activities of these receptor-based switches are only partially understood. In this paper, we sought to understand the mechanism that underlies the activity of the ANTXR2 anthrax toxin receptor-based switch that binds to domains 2 and 4 of the protective antigen (PA) toxin subunit. Receptor-binding restricts structural changes within the heptameric PA prepore that are required for pore conversion to an acidic endosomal compartment. The transfer crosssaturation (TCS) NMR approach was used to monitor changes in the heptameric PA-receptor contacts at different steps during prepore-to-pore conversion. These studies demonstrated that receptor contact with PA domain 2 is weakened prior to pore conversion, defining a novel intermediate in this pathway. Importantly, ANTXR2 remained bound to PA domain 4 following pore conversion, suggesting that the bound receptor might influence the structure and/or function of the newly formed pore. These studies provide new insights into the function of a receptor-based molecular switch that controls anthrax toxin entry into cells. - Cellular receptors can act as molecular switches that initiate conformational changes in microbial proteins required for cellular entry. Examples of such switches include an anthrax toxin receptor (described in detail below) as well as those for a number of viruses including HIV-1 and other retroviruses [1,2,3], measles virus [4], and herpesviruses [5]. The mechanisms by which these receptorbased switches function to promote cellular entry are only partially understood. In this report we set out to define the mechanism by which a receptor-based switch regulates anthrax toxin prepore-topore conversion. Anthrax toxin, the key virulence factor secreted by Bacillus anthracis, is a bacterial AB toxin composed of three independent, plasmid-encoded polypeptide chains: the receptor-binding (B) moiety, protective antigen (PA), and two different enzymatic (A) moieties, lethal factor (LF) and edema factor (EF) [6,7,8]. The first step in cellular intoxication involves binding of an 83 kD form of PA (PA83) to specific cell surface receptors. Although several PA receptors have been defined [9,10,11], anthrax toxin receptor type 2 (ANTXR2) (also known as capillary morphogenesis protein 2; CMG2), is the most physiologically relevant receptor [12,13,14]. ANTXR2 is a type 1 transmembrane protein and its extracellular von Willebrand factor type A (VWA) domain is the site of PAbinding [15,16]. Following receptor binding, PA83 is cleaved to a 63kD form (PA63) that spontaneously oligomerizes to form either a heptameric, or an octameric, PA63 prepore structure [17,18]. Oligomeric PA63-receptor complexes are then taken into cells primarily by a clathrin-dependent endocytic mechanism and delivered to an acidic endosomal compartment where low pH triggers formation of a PA63 pore within an endosomal membrane [19,20]. LF and EF are then translocated through the pore and delivered to the cytosol where they promote intoxication [21]. X-ray structural analysis of monomeric and heptameric PAANTXR2 VWA-domain complexes revealed that the receptor acts as a molecular switch or clamp that inhibits prepore-to-pore conversion at neutral pH [15,16]. Specifically, the receptor VWAdomain interacts with the base regions of PA domains 2 and 4, thereby sterically hindering the movement of the PA 2b3-2b4 loop region necessary for pore formation [15,16]. Those findings led to a model in which release of the receptor contact with PA domain 2 at an acidic endosomal pH is necessary to permit the conformational changes required for PA pore formation [15,16]. Consistent with this idea, the pH threshold of the receptorregulated toxin pore formation can be dictated by specific amino acid residues located at the PA domain 2-binding region of the ANTXR2 VWA-domain [22]. Presently, it is not clear if PA domain 2-receptor contacts are released at a step that occurs prior to, or is coincident with, prepore-to-pore conversion. Furthermore, it is not clear if the receptor remains attached following pore conversion and, if so, how it remains attached. Evidence supporting dissociation has come from co-immunoprecipitation experiments [23] and from previous NMR studies [24,25]. On the other hand, evidence in favor of receptor attachment has come from other co-immunoprecipitation studies [19,26], from NMR binding studies perThe bacterium that causes anthrax produces a toxin called anthrax toxin that is largely responsible for causing disease symptoms. The first step in anthrax intoxication involves binding of the toxin to a specific protein, called a receptor, on the cell surface. Receptor-binding acts like a switch to prevent the toxin from forming a pore in a cell membrane until the toxin-receptor complex is taken up into cells and delivered to a specific location (called an endosome) where it is exposed to an acid bath. This acidic environment promotes structural changes in the toxin leading to pore formation in the endosomal membrane. In this report, we have studied how the receptor regulates pore formation by following the associated changes in toxin-receptor contacts. These studies have defined a new toxin-receptor intermediate in the pathway leading to pore conversion and demonstrate that the receptor remains bound after pore conversion. Our results provide important new insights into how the receptor regulates anthrax toxin pore formation, information that could be useful for designing new therapeutic strategies to treat this disease. formed with a fragment (Domain 4) of PA [27], and from the finding that the presence of a receptor seems to influence voltagedependent inactivation and small molecule inhibition properties of the newly formed pore [28]. Based upon structural considerations, it has also been argued that the receptor may remain bound to serve as a structural support for the pore [16,29]. To clarify these issues, we have employed NMR techniques to monitor changes in the PA63 heptamer-ANTXR2 VWA domain contacts as a function of pH. Initially we attempted to examine the interaction between the ANTXR2 VWA domain and PA63 using chemical shift perturbation (CSP) by titratin (...truncated)