FUNCIONAMIENTO DEL COMEDOR ESCOLAR.- NORMAS DE CUMPLIMIENTO OBLIGATORIO

The cytotoxicity of pore-forming colicins, results from the formation of ion-

permeable channels formed in the inner membrane of the susceptible cell, by the C- terminal pore-forming domain of the colicin (Dankert et al 1982). Several models have been proposed to explain the formation of the channel, but none are readily accepted (Lakey et al 1993, van der Goot 1991, Zakharov and Cramer 2002). The initial interaction of the cytotoxic domain with the membrane is predicted to be electrostatic, as pore-forming colicins require an acidic pH for binding to artifical membrane vesicles (Davidson et al 1995, Zakharov and Cramer 2002). A group of positive charges on the surface of the domain is predicted to orient the domain on the membrane surface (Elkins et al 1997). Once the cytotoxic domain has bound to the

extension of the helices and insertion into the membrane (van der Goot et al 1991, Cramer et al 1995, Zakharov and Cramer 2002). The requirement for partial

unfolding of the pore-forming domain, as it moves from the aqueous to the membrane phase, has been inferred from the onset of channel activity and the decrease in

structure, as monitored by CD, as a function of pH (Schendel and Cramer 1995).

1.10.1.1. Structures of pore-forming domains

The structures of the pore-forming domains of colicins A (Parker et al 1989), E1 (Elkins et al 1997), N (Vetter et al 1998), B (Hilsenbeck et al 2004) and Ia (Wiener et

al 1997) show that they consist of a bundle of eight amphipathic helices burying two

hydrophobic helices. The average length of the helices is 13 residues, but

approximately 17-20 residues are required to span the membrane bilayer (Elkins et al 1997, Zakharov and Cramer 2002). However, the α-helical content of colicin E1 increases by ~25 % on pore formation, which may cause a sufficient extension to allow the helices to span the membrane (Elkins et al 1997, Zakharov et al 1998). The two longest helices (the hydrophobic helices, 8 and 9) form a hydrophobic helical hairpin at the core of the domain, which is predicted to provide a membrane anchor after the initial interaction of the protein with the membrane bilayer (Zakharov and Cramer 2002).

1.10.1.2. Nature of the channel

The cytotoxic domain of the pore-forming colicin remains tightly bound to the membrane once it has inserted into the membrane (Kd ~ 2-3 nM, Heymann et al 1996). From the permeability of the channel formed by colicin E1 to various probes, the diameter of the channel formed by colicin E1 has been estimated to be 9 Ǻ at the narrowest part (Bullock et al 1992). However, if the elongated shape of these probes is taken into account, the channel could be as small as 4-5 Ǻ (Cramer et al 1995). The size of the channel formed varies according to the colicin.

The channel activity of colicin A has been measured by analysing the kinetics of potassium efflux induced by the colicin (Bourdineaud et al 1990). The channels formed in vivo were shown to be voltage and pH dependent. After addition of colicin, the membrane potential drops to -85mV and the channel closes if the potential

voltage at which 50 % of channels are activated, varies for different colicins (Bénédetti and Géli 1996). The gating voltage for full-length colicins is pH

dependent but the gating voltage for the pore-forming domain is not pH dependent. This indicates that regions other than the pore-forming domain regulate channel behaviour (Bénédetti and Géli 1996).

The channel allows movement of monovalent ions (Na+_{, K}+ _{and Cl}-_{) with a single} channel conductance of > 106 _{ions channel}-1 _second-1_{, which is sufficient to allow a} single colicin pore to depolarise the membrane (Bénédetti and Géli 1996, Bullock et

al 1983). The depolarisation causes inhibition of active transport and depletion of

intracellular ATP, potassium and phosphate, leading to cell death (Elkins et al 1997).

1.10.2.

Endonucleases

1.10.2.1. DNases

The DNase colicins E2, E7, E8 and E9 cleave the host chromosomal DNA. They share a high sequence identity in their receptor binding and translocation domains but are only ~70-80 % identical in their DNase domains. X-ray crystallography and activity assays have localized the DNase domain of colicin E9 to residues 449-582 (Kleanthous et al 1999, Pommer et al 1998).

Structures of DNase domains

Structures of the DNase domains of colicins E7 and E9 with their respective immunity proteins have recently been determined (Ko et al 1999, Kleanthous et al 1999). The crystal structure for the E9 DNase-Im9 complex at 2.05Å resolution shows the DNase domain consists of a central core of β-sheet surrounded by α-helices (Kleanthous et al 1999). The C-terminal 32 amino acid residues show sequence identity to the HNH family of homing endonucleases (Shub et al 1994). The HNH motif resembles a distorted zinc finger and forms the core of the DNase active site (Pommer et al 1999). This is supported by site-directed mutagenesis experiments, which were used to identify putative active site residues in the DNase domain of colicin E9 (Garinot-Schneider et al 1996). Three single site mutations were

identified which completely destroyed the toxic action of the colicin, R544A, E548A and H575A. All three residues are highly conserved amongst the DNase colicins

the mechanism of the enzyme (Kleanthous et al 1999). A nickel ion is bound in the crystal structure, and NMR has confirmed that this ion is coordinated by three histidine side chains and a phosphate molecule (Hannan et al 2000).

The crystal structure of the colicin E7-Im7 complex at 2.3 Å resolution shows the DNase domain is a novel αβ protein with a Zn2+ _{ion bound to 3 histidine residues and} a water molecule and contains a zinc finger motif (Ko et al 1999). The three

catalytically important residues identified by site-directed mutagenesis of colicin E9 are conserved in colicin E7 and are located near the Zn2+ _ion.

Colicin E9 has been shown to share sequence homology with CAD enzymes ie DNases involved in degradation of chromatin during the terminal stages of apoptosis (Walker et al 2002). The similarity is supported by mutagenesis experiments. H263 in mouse CAD, equivalent to H103 in the colicin E9 DNase domain, is predicted to activate the hydrolytic water molecule. H308 of mouse CAD, equivalent to H127 in the colicin E9 DNase, is suggested to be the Mg2+ binding residue. The CAD enzymological properties are analogous to those of the colicin E9 DNase and therefore it is speculated that colicin DNases and CADs cleave DNA by the same mechanism, which is an interesting possibility given that both enzymes are involved in the degradation of chromosomal DNA, leading to cell death (Walker et al 2002).

Specificity of colicin E9 DNase

The colicin E9 DNase has been shown to preferentially nick double-stranded DNA at thymine bases, producing 3’-hydroxy and 5’-phosphate termini and it is suggested that a DNase monomer binds to the phosphate backbone of each strand (Pommer et al 2001). The DNase does not cleave mononucleotide phosphoryl esters or dinucleotide substrates. The DNase also cleaves single-stranded DNA, but the presence of a transition metal is required (Pommer et al 2001).

The metal ion

The role of the metal ion bound to the DNase domain is still debateable and it has been suggested that it may play a catalytic or structural role.

The metal-bound water molecule may serve as a nucleophilic hydroxide ion that attacks the phosphate atom (Ko et al 1999). A cleft near the three catalytically

DNase domain of colicin E7 has shown that although zinc ions are not required for binding of the domain to DNA, the metal ion is required for hydrolysis of DNA, further suggesting that the metal ion is involved in DNA hydrolysis (Ku et al 2002). Ordinarily the metal ion in the zinc finger motif would be tetrahedrally coordinated with four protein ligands. However, in the DNase domain of colicins E9 and E7, the fourth ligand is either a phosphate molecule or a water molecule respectively. This could reduce the affinity of the protein for the metal ion, allowing removal of the metal ion at some point during translocation to allow partial unfolding (Pommer et al 1999). Therefore, the metal ion could play another role in stabilising the DNase domain in the extracellular environment.

Although zinc or nickel have been identified in the crystal structures of colicin E7 and E9, enzymological characterisation of the colicin E9 DNase domain showed that it can use a number of metal ions to cleave DNA, the identity of which governs whether the enzyme produces single or double strand breaks (Pommer et al 1998).

Although zinc can be used for hydrolysis of DNA by colicin E7, zinc does not support hydrolysis of any DNA substrate by colicin E9 (Pommer et al 2001). The

physiological metal ion for colicin E9 has been shown to be magnesium, which allows the formation of double-strand breaks in the DNA (Walker et al 2002).

1.10.2.2. RNases

The cytotoxic domains of the RNase colicins display a greater sequence conservation (~80-90%) than that for DNase colicins.

Colicin E3, E4, E5 and E6 kill cells by inactivating the protein biosynthetic machinery (Nomura and Witten 1967).

rRNases

Colicin E3 is a 58 kDa ribonuclease that cleaves 16S ribosomal RNA (rRNA) at the 49th _{phosphodiester bond from the 3’ end (Bowman et al 1971). This bond is at a} critical position near where interactions of the A site tRNA and the mRNA on the ribosome occur (Cate et al 1999). The crystal structure of the cytotoxic domain of colicin E3 in complex with its immunity protein has been solved to 2.4Å resolution revealing a highly twisted central antiparallel β-sheet elaborated with a short N- terminal helix (Carr et al 2000b, Soelaiman et al 2001). This fold is significantly

with molecular modelling and mutagenesis has revealed a putative active site within the toxin containing a His-Glu catalytic pair (Soelaiman et al 2001, Walker et al 2004).

Docking of the crystal structure of the RNase domain of colicin E3 onto the crystal structure of the 30S ribosomal subunit, has lead to the proposal of a catalytic mechanism with residues E517, H513 and D510 making up the catalytic triad (Zarivach et al 2002). Residue R545 is likely to stabilise the negatively charged penta-coordinated cyclic phosphate atom transition state. Alanine mutagenesis confirmed the importance of residue H513 and E517 as the acid-base pair in catalysis (Walker et al 2004).

A comparison of the sequences of the C-terminal domains of colicins E4 and E6 with colicin E3 shows that colicins E4 and E6 are E3 homologs and also cleave the 16S rRNA. However, the C-terminal region of E5 exhibits no sequence similarity to colicin E3.

tRNases

Examination of colicin E5 activity in vitro and in vivo demonstrated that the target of colicin E5 is not ribosomes, as in the case of colicin E3, but tRNA for tyrosine, histidine, asparagine and aspartate. These tRNAs all contain the guanine analog, queuine, at the wobble position of each anticodon, although the sequence with an unmodified guanine instead of queuine is also sensitive to the colicin (Ogawa et al 1999, Masaki and Ogawa 2002). Colicin E5 hydrolyses these tRNAs on the 3’ side of this nucleotide. Colicin D has also been shown to act as a tRNase, cleaving arginine tRNAs (Masaki and Ogawa 2002). The tRNase activity of colicin D has been localised to the C-terminal 91 residues (de Zamaroczy et al 2001).