The crystal structure of colicin IA

Abstract

The structure of the entire chmmel-fonning bacte1iocin (protein toxin) colicin Iahas been solved to a resolution of2.4A by multiple isomorphous replacement. The fonnation of ion-penneable chmmels in ta.rget cell membrm1es is a general mechanism of cytotoxicity. The process involves secretion of a soluble protein which inserts into the plasma membrm1e of the target cell m1d fon11S a lethal pore. Colicins, Escherichia coli protein toxins, are a well-characterized exmnple of this class of proteins. Colicin Ia crystals, comprised of approximately 80% solvent are in spacegroup C222 1 (a=64.4A, b=l78.6A, c=285.5A). All data sets used in the structure determination were collected fi·om fi·ozen crystals with a synchrotr·on light source (SSRL bean1line 7-1 ). Heavy atom de1ivatives were obtained using mercmial soaks of engineered single-site cysteine mutants.The str1.1cture of the 69kD colicin Ia protein reveals the structural relationships between the three distinct domains which function, respectively, to i) bind to a receptor on the outer membrane of susceptible bacte1ia, ii) tr·anslocate across the outer membrane tlu·ough the receptor, and iii) bind to the inner membrane and fom1 a pore in the presence of the transmembrane voltage. The domains are sepm·ated by an extraordinmily long helical coiled-coil. The cqstal structure of the a-;sembly domain of the cmtilage oligomeric matrix protein (COMP), a pentameric glycoprotein of the thrombospondin family found in cmtilage m1d tendon, was detem1ined at 2.03 A resolution using MIRAS phasing with xenon, (CH3)3Pb(COOCH3)3 and Pr(COOCH3)3 fmther improved by solvent flattenincr and five~fold avera£ing. Self-association ofCOMP, as well as of at le~t two other extracellul~ matrix proteins, thrombosponilins 3 and 4, is achieved through the fonmtion of a five-str·m1ded a-helical bundle which involves 64 N-tem1inal residues (20-83). The complex is fiuther stabilized by the interchain disulphide bonds between cysteines 68 m1d 71. Circular dichroism measurements show that the structme of the assembly domain remains intact even at temperatures above I OOOC. Wlllie the cryst£'11 structures of two-, tlu·ee-and fom-stranded a-helical bm1dles were reported before, that one of tl1e pentai11eric coiled coil is novel. The migins of the extreme thennal stability, the unusual degree of oligomelization and tl1e role of the intemal hydrophobic axial cavity are tl1e questions to be addressed in tl1e cmTent study. The peptides containing 64, 52 or 46 residues were produced by expression in Escherichia coli, but well diffracting crystals were obt£1ined only witl1 t11e 46 residues fragment (Ph a=38.47 A, b=49.47 A, c= 54.98 A and ~= 103.84°). The centr·al pmt of tl1e molecule w11ich includes five heptad repeats (residues 29-65), obeys approximate five-fold symmetry while the remaining residues at theN-and C-tem1ini show significant deviations fi·om tl1at. Str·ong sy1m11etry violations could explain tl1e little success acl1ieved in om earlier attempts to solve t11e structure by tl1e molecuhu·replacem...

Colicin Biology

“…This implies that the three domains are linked via flexible regions that are subject to large-scale structural modifications. This is indeed the case, since most colicins (except colicins N and B) showed elongated structures with extended coiled-coil ␣-helices (102,607,675) and have been shown to unfold during the translocation process (24). Based on calorimetric, spectroscopic, and crystallographic experiments, it has been proposed that colicin E1 unfolds by a process triggered by binding to the BtuB receptor (237,371).…”

“…They are elongated proteins (358) and are hard to crystallize. The crystal structure of the C-terminal domain of colicin A was obtained in 1989 (502), long before that of an entire colicin was obtained, which did not appear until 1997, when the structure of colicin Ia was solved (675). This has now been joined by several other colicin structures (177,267,607,650).…”

Colicin E2 Is Still in Contact with Its Receptor and Import Machinery When Its Nuclease Domain Enters the Cytoplasm

“…The two arms of the Y shape correspond to the N-terminal domain and the C-terminal domain of colicins, whereas the stem corresponds to the central domain. The two arms are long enough to allow the C-terminal domain and the N-terminal domain to interact with the inner membrane, while the central domain remains attached to the outer membrane (32,36). It is difficult to evaluate the time required for DNase colicins to kill sensitive cells, because there is no rapid and sensitive assay to measure DNA damage.…”

The Crystal Structure of the Lipid II-degrading Bacteriocin Syringacin M Suggests Unexpected Evolutionary Relationships between Colicin M-like Bacteriocins

“…Although it is worth considering that the structurally analogous receptor binding domains of colicin or syringacin M may have evolved through convergent evolution, it seems unlikely that such structural homology would have arisen independently, given the structural diversity of receptor binding domains from other colicins, which essentially share the same function (38,48,49). Second, structural conservation is unlikely to have evolved in order to stabilize the catalytic domain or overall structure of colicin M-like bacteriocins because the receptor binding domains of pectocin M1 and M2 consist of a ferredoxin-like protein with no structural similarity to the receptor binding domains of colicin or syringacin M (8).…”