Colicin crystal structures: pathways and mechanisms for colicin insertion into membranes

The X-ray structures of the channel-forming colicins Ia and N, and endoribonucleolytic colicin E3, as well as of the channel domains of colicins A and E1, and spectroscopic and calorimetric data for intact colicin E1, are discussed in the context of the mechanisms and pathways by which colicins are imported into cells. The extensive helical coiled-coil in the R domain and internal hydrophobic hairpin in the C domain are important features relevant to colicin import and channel formation. The concept of outer membrane translocation mediated by two receptors, one mainly used for initial binding and second for translocation, such as BtuB and TolC, respectively, is discussed. Helix elongation and conformational flexibility are prerequisites for import of soluble toxin-like proteins into membranes. Helix elongation contradicts suggestions that the colicin import involves a molten globule intermediate. The nature of the open-channel structure is discussed.     

Computational studies of colicin insertion into membranes: the closed state

Colicins are water-soluble toxins that, upon interaction with membranes, undergo a conformational change, insert, and form pores in them. Pore formation activity is localized in a bundle of 10 α-helices named the pore-forming domain (PFD). There is evidence that colicins attach to the membrane via a hydrophobic hairpin embedded in the core of the PFD. Two main models have been suggested for the membrane-bound state: penknife and umbrella, differing in regard to the orientation of the hydrophobic hairpin with respect to the membrane. The arrangement of the amphipathic helices has been described as either a compact three-dimensional structure or a two-dimensional array of loosely interacting helices on the membrane surface. Using molecular dynamics simulations with an implicit membrane model, we studied the structure and stability of the conformations proposed earlier for four colicins. We find that colicins are initially driven towards the membrane by electrostatic interactions between basic residues and the negatively charged membrane surface. They do not have a unique binding orientation, but in the predominant orientations the central hydrophobic hairpin is parallel to the membrane. In the inserted state, the estimated free energy tends to be lower for the compact arrangements of the amphipathic helix, but the more expanded ones are in better agreement with experimental distance distributions. The difference in energy between penknife and umbrella conformations is small enough for equilibrium to exist between them. Elongation of the hydrophobic hairpin helices and membrane thinning were found unable to produce stabilization of the transmembrane configuration of the hydrophobic hairpin.     

Colicin S8 export: extracellular and cytoplasmic colicin are different

The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process.     

Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import

The interaction of colicins with target cells is a paradigm for protein import. To enter cells, bactericidal colicins parasitize Escherichia coli outer membrane receptors whose physiological purpose is the import of essential metabolites. Colicins E1 and E3 initially bind to the BtuB receptor, whose beta-barrel pore is occluded by an N-terminal globular "plug". The x-ray structure of a complex of BtuB with the coiled-coil BtuB-binding domain of colicin E3 did not reveal displacement of the BtuB plug that would allow passage of the colicin (Kurisu, G., S. D. Zakharov, M. V. Zhalnina, S. Bano, V. Y. Eroukova, T. I. Rokitskaya, Y. N. Antonenko, M. C. Wiener, and W. A. Cramer. 2003. Nat. Struct. Biol. 10:948-954). This correlates with the inability of BtuB to form ion channels in planar bilayers, shown in this work, suggesting that an additional outer membrane protein(s) is required for colicin import across the outer membrane. The identity and interaction properties of this OMP were analyzed in planar bilayer experiments.OmpF and TolC channels in planar bilayers were occluded by colicins E3 and E1, respectively, from the trans-side of the membrane. Occlusion was dependent upon a cis-negative transmembrane potential. A positive potential reversibly opened OmpF and TolC channels. Colicin N, which uses only OmpF for entry, occludes OmpF in planar bilayers with the same orientation constraints as colicins E1 and E3. The OmpF recognition sites of colicins E3 and N, and the TolC recognition site of colicin E1, were found to reside in the N-terminal translocation domains. These data are considered in the context of a two-receptor translocon model for colicin entry into cells.     

BID-dependent and BID-independent pathways for BAX insertion into mitochondria

In the absence of an apoptotic signal, BAX adopts a conformation that constrains the protein from integrating into mitochondrial membranes. Here, we show that caspases, including caspase-8, can initiate BAX insertion into mitochondria in vivo and in vitro. The cleavage product of caspase-8, tBID, induced insertion of BAX into mitochondria in vivo, and reconstitution in vitro showed that tBID, either directly or indirectly, relieved inhibition of the BAX transmembrane signal-anchor by the NH2-terminal domain, resulting in integration of BAX into mitochondrial membrane. In contrast to these findings, however, Bid-null mouse embryo fibroblasts supported Bax insertion into mitochondria in response to death signaling by either TNFalpha or E1A, despite the fact that cytochrome c release from the organelle was inhibited. We conclude, therefore, that a parallel Bid-independent pathway exists in these cells for mitochondrial insertion of Bax and that, in the absence of Bid, cytochrome c release can be uncoupled from Bax membrane insertion.     

High-resolution crystal structure of a truncated ColE7 translocation domain: implications for colicin transport across membranes

ColE7 is a nuclease-type colicin released from Escherichia coli to kill sensitive bacterial cells by degrading the nucleic acid molecules in their cytoplasm. ColE7 is classified as one of the group A colicins, since the N-terminal translocation domain (T-domain) of the nuclease-type colicins interact with specific membrane-bound or periplasmic Tol proteins during protein import. Here, we show that if the N-terminal tail of ColE7 is deleted, ColE7 (residues 63-576) loses its bactericidal activity against E.coli. Moreover, TolB protein interacts directly with the T-domain of ColE7 (residues 1-316), but not with the N-terminal deleted T-domain (residues 60-316), as detected by co-immunoprecipitation experiments, confirming that the N-terminal tail is required for ColE7 interactions with TolB. The crystal structure of the N-terminal tail deleted ColE7 T-domain was determined by the multi-wavelength anomalous dispersion method at a resolution of 1.7 angstroms. The structure of the ColE7 T-domain superimposes well with the T-domain of ColE3 and TR-domain of ColB, a group A Tol-dependent colicin and a group B TonB-dependent colicin, respectively. The structural resemblance of group A and B colicins implies that the two groups of colicins may share a mechanistic connection during cellular import.     

On the insertion of proteins into membranes

Recent data concerning the primary structure and the interactions of proteins with membranes suggest the existence of two classes of integral membrane proteins. In the first class, the polypeptide chain crosses the membrane only once. The membrane penetrating fragment is markedly hydrophobic and contains several positive charges on its C-terminal border. In the second class, the protein is folded in a complex fashion within the membrane and the knowledge of its amino acid sequence is not sufficient to predict the manner in which the protein interacts with the membrane.     

Herpesvirus glycoprotein synthesis and insertion into plasma membranes.

In the presence of the antibiotic tunicamycin (TM), glycosylation of herpes simplex virus glycoproteins is inhibited and non-glycosylated polypeptides analogous to the glycoproteins are synthesized (Pizer et al., J. Virol. 34:142-153, 1980). The synthesis of viral proteins and DNA occurs in TM-treated cells. By electron microscopy, nucleocapsids can be observed both in the nucleus and the cytoplasm of TM-treated cells; a small number of enveloped virions were observed on the cell surface. Analyses of the proteins in partially purified virus readily detects viral glycoproteins in the control cells, but neither glycoproteins nor nonglycosylated polypeptide analogs were observed in the virus prepared from TM-treated cells. By labeling the surface of infected cells with 125I, viral glycoproteins were detected as soon as 90 min after infection even when protein synthesis was inhibited with cycloheximide and glycosylation was blocked with TM. Labeling the proteins synthesized in infected cells with [35S]methionine showed that the surface glycoproteins detected in the cycloheximide- and TM-treated cells were not synthesized de novo after infection, but were placed on the cell surface by the infecting virus. Studies with metabolic inhibitors and a temperature-sensitive mutant blocked early in the infectious cycle showed that glycoproteins gA/gB and gD were synthesized soon after infection, but that the synthesis of gC was delayed. Under conditions of infection, in which gC and its precursor pgC are not produced, we have been able to observe the relationships between the glycosylated polypeptides that correspond to pgA/pgB and the nonglycosylated analog made in the presence of TM.     

Colicin import into E. coli cells: A model system for insights into the import mechanisms of bacteriocins

Bacteriocins are a diverse group of ribosomally synthesized protein antibiotics produced by most bacteria. They range from small lanthipeptides produced by lactic acid bacteria to much larger multi domain proteins of Gram negative bacteria such as the colicins from Escherichia coli. For activity bacteriocins must be released from the producing cell and then bind to the surface of a sensitive cell to instigate the import process leading to cell death. For over 50 years, colicins have provided a working platform for elucidating the structure/function studies of bacteriocin import and modes of action. An understanding of the processes that contribute to the delivery of a colicin molecule across two lipid membranes of the cell envelope has advanced our knowledge of protein–protein interactions (PPI), protein–lipid interactions and the role of order–disorder transitions of protein domains pertinent to protein transport. In this review, we provide an overview of the arrangement of genes that controls the synthesis and release of the mature protein. We examine the uptake processes of colicins from initial binding and sequestration of binding partners to crossing of the outer membrane, and then discuss the translocation of colicins through the cell periplasm and across the inner membrane to their cytotoxic site of action. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Biosynthesis and insertion of Wolfgram protein into optic nerve membranes

Antibodies against pig brain Wolfgram protein (WP) were prepared and utilized in the analysis of WP biosynthesis in membranes from optic nerves of 20 day-old rats. Newly synthesized WP appeared rapidly (<5 min) in myelin and in a non-myelin microsome fraction and accumulated in both thereafter. Monensin did not affect the insertion of WP in either membrane fraction. These results are consistent with biosynthesis of WP on free ribosomes.     

Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate     

Catalyzed insertion of proteins into phospholipid membranes: specificity of the process

The process of insertion of intrinsic proteins into phospholipid membranes conjures up the thought of enormous energy barriers but is a routine occurrence in cells. Proteinaceous complexes responsible for protein targeting/translocation/insertion into membranes have been studied intensively. However, the mitochondrial voltage-dependent anion channel (VDAC), can insert into phospholipid membranes by an auto-catalytic process called "auto-directed insertion." This process results in an oriented insertion of VDAC channels and an increase in insertion rate per unit area of 10 orders of magnitude. Here we report that VDAC catalyzes the insertion of PorA/C1 and KcsA by increasing their calculated insertion rate per unit area by 9 orders of magnitude with no detectable effect on the insertion of alpha-hemolysin. This was measured as a reduction in the delay before the first insertion of these proteins. Gramicidin and PorA/C1 accelerate the calculated insertion rate per unit area of VDAC by 8 and 9 orders of magnitude, respectively. Only PorA/C1 increases the overall rate of VDAC insertion (50-fold) over the self-catalyzed rate. Our results indicate that catalyzed insertion of proteins into phospholipid membranes does not arise simply from disturbance of the phospholipid membrane because it shows strong specificity.     

A mechanism for the partial insertion of protein kinase C into membranes

We propose that the principle driving force allowing protein kinase C (PKC) to insert partway into membranes is the transient creation of an interior hydrophilic phase within the membrane. We further suggest that this phase is composed of non-bilayer-forming elements, such as diacylglycerol or phorbol esters. We used the combination of fluorescence resonance energy transfer (using fluorescently labeled phospholipid molecules and the endogenous tryptophan residues of PKC) and fluorescence quenching by the water-soluble reagent potassium iodide. The experimental system used micelles and purified PKC. Our model accounts for both the established kinetic data on PKC as well as the physical requirements of protein-membrane interaction. Moreover, it establishes PKC as the first example of a partially embedded membrane protein, and provides a mechanism to account for its activation.     

Colicin import and pore formation: a system for studying protein transport across membranes?

Pore-forming colicins are a family of protein toxins (M_r40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage-gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient-uptake systems and the so-called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage-gated channels). The Tol-dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α-helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin-resistant mutant of OmpF. The X-ray crystal structure of the channel-forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site-directed muta-genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N-terminal domains of colicins interact with the lumen of the OmpF pore and then with the C-terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C-terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.     

Colicins and bacterial membranes: structures and functions. II. Studies on reconstituted homologous and hybrid membranes prepared from cytoplasmic membranes of untreated and colicin K-treated bacteria.

Cytoplasmic membranes of Escherichia coli K12 C600 treated and not treated with colicin K were dissociated into unsolubilized and solubilized fractions. Neither fraction catalyzed ATP-linked transhydrogenase activity. Mixtures of unsolubilized fractions of the untreated bacteria with solubilized fractions of either the treated or untreated bacteria yielded reconstituted membranes with restored ATP-linked transhydrogenase activity. The level of the activity was similar to that of the undissociated membranes of untreated bacteria. The membranes which were reconstituted from unsolubilized fractions of the treated bacteria and the solubilized fraction of the treated or the untreated bacteria showed impairment of activity. The impairment is not due to an inability to bind ATPase of the soluble fraction or to an incorrect binding of the ATPase. The impaired, reconstituted membranes showed striking decreases in the relative amounts of three proteins with apparent molecular weights of 122,000, 73,000, and 62,000. The affected proteins were found to be components of the unsolubilized membrane fraction. It is, thus, concluded that the impaired activity is due to the defective nature of the unsolubilized membrane fraction of colicin-treated cells.     

Infliximab crystal structures reveal insights into self-association

Aggregation and self-association in protein-based biotherapeutics are critical quality attributes that are tightly controlled by the manufacturing process. Aggregates have the potential to elicit immune reactions, including neutralizing anti-drug antibodies, which can diminish the drug's efficacy upon subsequent dosing. The structural basis of reversible self-association, a form of non-covalent aggregation in the native state, is only beginning to emerge for many biologics and is often unique to a given molecule. In the present study, crystal structures of the infliximab (Remicade) Fc and Fab domains were determined. The Fab domain structures are the first to be reported in the absence of the antigen (i.e., tumor necrosis factor), and are consistent with a mostly rigid complementarity-determining region loop structure and rotational flexibility between variable and constant regions. A potential self-association interface is conserved in two distinct crystal forms of the Fab domain, and solution studies further demonstrate that reversible self-association of infliximab is mediated by the Fab domain. The crystal structures and corresponding solution studies help rationalize the propensity for infliximab to self-associate and provide insights for the design of improved control strategies in biotherapeutics development.