Structural Studies of Adenovirus Type‐2 Hexon Protein

Abstract

The adenovirus type 2 hexon protein produced in excess during infection was carboxymethylated by reduction and alkylation with iodo[2-14C]acetate in 6 M guanidine hydrochloride. Only one type of polypeptide chain was detected by exclusion chromatography in dissociating buffers. Peptide mapping experiments, sequence analysis of peptides and identification of possible protein terminal residues also suggest that all subunits are similar and probably identical.Structural analysis of ['4C]carboxymethylated hexon and of hexon which was labeled in vivo with [3SS]cysteine revealed six unique cysteine derivatives in soluble tryptic peptides. A seventh unique cysteine derivative, only partly carboxymethylated, was present in the tryptic core material and was recovered after chymotryptic digestion. Seven unique cysteine derivatives in the hexon subunit, correspond to a minimum molecular weight of about 100000 for the subunit. This value is consistent with values obtained from exclusion chromatography in guanidine hydrochloride, from mercurial titration of an accessible thiol group in the intact protein and from peptide mapping experiments.Peptides corresponding to more than one tenth of the total number of residues in the protein chain were characterized and, with the inclusion of other peptides studied, about one quarter of all residues are accounted for. The results exclude extensive regions of identity in primary structure within the hexon subunits and the partial resistance towards attack by proteolytic enzymes is structurally explained.The adenoviruses are medium-sized icosahedral DNA viruses, some of which are oncogenic. The hexon is the main capsomer, occurring at 240 non-vertex positions in the capsid and contains one of at least ten [1,2] viron polypeptides. It was the first animalvirus protein to be crystallized [3]. It is soluble in native form and is produced in excess during viral infection. Widely different molecular weights have been reported for the intact hexon as well as for its subunits (mainly within values of 200 000 -400 000, and 60000-120000, respectively, for a review, see [4]).The hexon subunit thus contains a large polypeptide, the maximum value of which accounts for the coding capacity of about 11 % of the entire viral genome.Recent analysis of sedimentation velocity in the absence or presence of guanidine-HC1, of equilibrium sedimentation, of crystallographic parameters, andAbbreviations. Tos-PheCH,Cl, L-l-tosylamido-2-phenylethyl chloromethyl ketone; Tos-LysCH,Cl, l-chloro-3-tosylamido-7-amino-2-heptanone; dansyl, S-dimethylaminonaphthalene-l-sulphonyl.Enzymes. Chymotrypsin (EC 3.4.21.1); trypsin (EC 3.4.21.4); thermolysin (EC 3.4.24.4); staphylococcal protease (EC 3.4.99 [1,6,7] for the subunit. Crystallographic studies also reveal three asymmetric units in the hexon [4,8], which is consistent with results from electron microscopy [9]. Crystallographic data, however, also suggest additional elements of partial two-fold symmetry [lo] (and P.-E. Werner, personal communication) and an earlier ...

Limited Proteolysis and a Reactive Cysteine Residue Define Accessible Regions in the Native Conformation of the Adenovirus Hexon Protein

“…Data for this peptide are given in Table 2. The amino acid sequence of this peptide defines the labelled residue as one of the previously identified cysteine residues [4], which in the tentative sequence of the whole protein [l] is found at a region around position 670 (k 45 residues, depending on the order of other peptides). The labelled peptide accounts for over half of the incorporated radioactivity in the protein, although background labelling was present.…”

“…Soluble hexon produced in excess was purified by DEAE-cellulose chromatography and crystallization, as previously described [4]. Trypsin (treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained from Worthington Biochemical Corporation (Freehold, NJ, U.S.A.); chymotrypsin (treated with 1 -chloro-3-tosylamido-7-amino-2-heptanone) from Merck (Darmstadt, F.R.G.…”

“…Hexon prepared in the absence of thiols was reduced with dithiothreitol as indicated in Materials and Methods, in order to generate accessible cysteine residues [4]. Different samples were then carboxymethylated directly, or after dialysis to lower the amount of dithiothreitol.…”

“…Cysteine thiol groups have previously been titrated in the hexon [4,5]. It was then discovered that in the native conformation the equivalent of one protein thiol group per subunit is accessible to the reagents 2-chloromercuri-4-nitrophenol or 5,5'-dithiobis(2-nitrobenzoic acid), although further groups may react under certain conditions or after denaturation with dodecyl sulfate.…”

“…It was then discovered that in the native conformation the equivalent of one protein thiol group per subunit is accessible to the reagents 2-chloromercuri-4-nitrophenol or 5,5'-dithiobis(2-nitrobenzoic acid), although further groups may react under certain conditions or after denaturation with dodecyl sulfate. The accessible groups appear fully available only if the protein has been either protected from oxidation during preparation, or later reduced with thiols [4]. In the present work, accessible cysteine residues were modified by selective carboxymethylation with iodo[14C]acetate.…”