The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "φ-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the φ clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.Anthrax toxin is composed of three nontoxic proteins, which combine on eukaryotic cell surfaces to form toxic, noncovalent complexes. [See (1) for a review.] Protective antigen (PA), the protein translocase component, binds to a cellular receptor and is activated by a furin-family protease. The resulting 63-kD receptor-bound fragment, PA 63 , self-assembles into the prepore, which is a ring-shaped homoheptamer (Fig. 1A). The prepore then forms complexes with the two ~90-kD enzymatic components, lethal factor (LF) and edema factor (EF). These complexes are endocytosed and delivered to an acidic compartment (2). There, the prepore undergoes an acidic pH-dependent conformational rearrangement (3) to form an ion-conducting, cationselective, transmembrane pore (4), allowing bound LF and EF to translocate into the cytosol.The PA 63 pore (Fig. 1B) is believed to consist of a mushroom-shaped structure, with a globular cap connected to a β-barrel stem that is ~100 Å long (5, 6). A model of the 14-strand β barrel reveals its lumen, which is ~15 Å wide and can only accommodate structure as wide as an α helix (7). The narrow pore creates a structural bottleneck, requiring that the catalytic factors, LF and EF, unfold in order to be translocated (8, 9). The destabilization energy required to unfold the tertiary structure of LF and EF originates partly from the acidic pH in endosomes, which causes their N-terminal domains (LF N and EF N ) to become molten globules (MG) (7). A positive membrane potential [+Δψ (10)], when coupled with these acidic pH conditions, is sufficient to drive LF N through PA 63 pores formed in planar lipid bilayers (9). To enter the narrow confines of the ~15-Å-wide lumen, LF N must shed its residual tertiary structure and convert from the MG form to an extended, "translocatable" conformation (7). How does a solvent-filled pore mediate the disassembly of an MG protein, packed, albeit loosely, with hydrophobically dense stretches of polypeptide? We surmised that an interaction surface inside the pore might facilitate further unfolding of the MG to the extended, translocatable form. †To whom correspondence should be addressed.

After binding to cellular receptors and proteolytic activation, the protective antigen component of anthrax toxin forms a heptameric prepore. The prepore later undergoes pH-dependent conversion to a pore, mediating translocation of the edema and lethal factors to the cytosol. We describe structures of the prepore (3.6 Å) and a prepore:receptor complex (4.3 Å) that reveal the location of poreforming loops and an unexpected interaction of the receptor with the pore-forming domain. Lower pH is required for prepore-topore conversion in the presence of the receptor, indicating that this interaction regulates pH-dependent pore formation. We present an example of a receptor negatively regulating pH-dependent membrane insertion.M any bacteria that colonize mammalian hosts have evolved mechanisms for introducing bacterial enzymes into the cytosolic compartment of host cells. These enzymes disrupt metabolism in various ways, disabling professional phagocytes and͞or other cells of the host's immune system. Bacillus anthracis accomplishes this disruption by secreting a tripartite toxinanthrax toxin, consisting of two intracellularly acting enzymes together with a multifunctional protein that delivers the enzymes to the cytosol (1). The two enzymes are: edema factor (EF, 89 kDa), an adenylate cyclase (2), and lethal factor (LF, 90 kDa), a metalloprotease specific for mitogen-activated protein kinase kinases (3, 4). The delivery component, termed protective antigen (PA, 83 kDa), is a receptor-binding protein that forms a pore in the endosomal membrane, enabling EF and LF to cross to the cytosol.PA, EF, and LF combine at the surface of receptor-bearing cells to form a series of toxic noncovalent complexes (1). PA is a four-domain molecule that binds to either of two cell-surface receptors, capillary morphogenesis protein 2 (CMG2) or anthrax toxin receptor͞tumor endothelial marker 8 (ATR͞TEM8) (5-7). Proteolysis at a furin-sensitive cleavage site within domain 1 (residues 1-258) removes a 20-kDa fragment, PA 20 , from the N terminus (8), leaving a 63-kDa fragment, PA 63 , bound to the receptor. The remaining part of domain 1 (residues 168-258) forms the N terminus of PA 63 and functions in oligomerization and in binding EF and LF (Fig. 1a). In the absence of PA 20 , PA 63 self-associates to form a ring-shaped heptamer (9), termed the prepore, the structure of which was previously determined at 4.5 Å (5). Domain 2 (residues 259-487) has a ␤-barrel core structure and lines the lumen of the heptamer (Fig. 1 a and b). There is a large amphipathic loop between strands 2␤2 and 2␤3 (residues 302-323) that is disordered in the crystal structure of monomeric PA and is believed to insert into the membrane as a hairpin to generate a 14-stranded ␤-barrel pore (10) (Fig. 1a). For this loop to reach and span the membrane, the 2␤2 and 2␤3 strands are predicted to peel away from the domain 2 core and, together with 2␤1, 2␤4, and the amphipathic hairpin, form an extended ␤-barrel involving residues 285-340 (5, 10, 11). Domain 3 (residues 488-59...

Therapeutic enzyme treatment disrupts Pseudomonas biofilms, potentiating antibiotics and ameliorating the innate immune system.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

BackgroundAmyloid precursor protein (APP) is enzymatically cleaved by γ-secretase to form two peptide products, either Aβ40 or the more neurotoxic Aβ42. The Aβ42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD). The transmembrane domain (TM) of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains.ResultsUsing synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers.ConclusionThe dissociation constants are correlated to both the Aβ42/Aβ40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Aβ production and Aβ42/Aβ40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and γ-secretase components, potentially revealing a new class of γ-secretase inhibitors.

Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXG-VXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and ϳ18 Å in structural space), work in concert to control TM-TM affinity synergistically.After their biosynthesis and subsequent integration into a membrane, many transmembrane (TM) 1 helices associate with other pre-formed helices to form functional membrane protein domains (1). Specificity for a given helix-helix interaction is achieved through the appropriate presentation of complementary side chains, which serve as recognition elements between associating helices. The most highly studied, and apparently widespread, mode of association is mediated by the so-called GXXXG motif, which is known to act as a universal scaffold for the assembly of both TM helices (2-9) and soluble ␣-helices (10). The GXXXG motif, where two glycine residues are separated by any three amino acids on a helical framework, gives rise to a flat surface region on one face of the helix. This arrangement of Gly residues permits the close approach of interacting helices, whereupon extensive packing interactions take place between pairs of surrounding residues. It has been proposed that a portion of the interactive strength of GXXXGmediated associations may originate from inter-helix hydrogen bonds between C␣ hydrogens and carbonyl oxygen atoms on the adjacent helix (11).The glycophorin A transmembrane (GpA-TM) segment is a well characterized transmembrane helix dimer that associates with high affinity, principally by using a central GXXXG motif (3,12). The details of side chain-side chain packing for GpA are known in considerable detail, having been gleaned originally from extensive mutagenesis experiments (3), computer modeling (13, 14) and, subsequently, from a high-resolution structural analysis using nuclear magnetic resonance (NMR) for the GpA dimer in both detergent micelles (12) and lipid bilayers (15).Despite the high occurrence of the GXXXG motif in transmembrane helices (7), GpA-TM is the only GXXXG peptide dimer with a structure det...

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.

Targeted degradation approaches such as proteolysis targeting chimeras (PROTACs) offer new ways to address disease through tackling challenging targets and with greater potency, efficacy, and specificity over traditional approaches. However, identification of high-affinity ligands to serve as PROTAC starting points remains challenging. As a complementary approach, we describe a class of molecules termed biological PROTACs (bioPROTACs)—engineered intracellular proteins consisting of a target-binding domain directly fused to an E3 ubiquitin ligase. Using GFP-tagged proteins as model substrates, we show that there is considerable flexibility in both the choice of substrate binders (binding positions, scaffold-class) and the E3 ligases. We then identified a highly effective bioPROTAC against an oncology target, proliferating cell nuclear antigen (PCNA) to elicit rapid and robust PCNA degradation and associated effects on DNA synthesis and cell cycle progression. Overall, bioPROTACs are powerful tools for interrogating degradation approaches, target biology, and potentially for making therapeutic impacts.