A census of glutamine/asparagine-rich regions: implications for their conserved function and the prediction of novel prions.

Glutamine/asparagine (Q/N)-rich domains have a high propensity to form self-propagating amyloid fibrils. This phenomenon underlies both prion-based inheritance in yeast and aggregation of a number of proteins involved in human neurodegenerative diseases. To examine the prevalence of this phenomenon, complete proteomic sequences of 31 organisms and several incomplete proteomic sequences were examined for Q/N-rich regions. We found that Q/N-rich regions are essentially absent from the thermophilic bacterial and archaeal proteomes. Moreover, the average Q/N content of the proteins in these organisms is markedly lower than in mesophilic bacteria and eukaryotes. Mesophilic bacterial proteomes contain a small number (0-4) of proteins with Q/N-rich regions. Remarkably, Q/N-rich domains are found in a much larger number of eukaryotic proteins (107-472 per proteome) with diverse biochemical functions. Analyses of these regions argue they have been evolutionarily selected perhaps as modular "polar zipper" protein-protein interaction domains. These data also provide a large pool of potential novel prion-forming proteins, two of which have recently been shown to behave as prions in yeast, thus suggesting that aggregation or prion-like regulation of protein function may be a normal regulatory process for many eukaryotic proteins with a wide variety of functions.

GroEL-GroES-mediated protein folding requires an intact central cavity.

The chaperonin GroEL is an oligomeric double ring structure that, together with the cochaperonin GroES, assists protein folding. Biochemical analyses indicate that folding occurs in a cis ternary complex in which substrate is sequestered within the GroEL central cavity underneath GroES. Recently, however, studies of GroEL "minichaperones" containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. Here we compare directly the folding activity of minichaperones with that of the full GroEL-GroES system. In agreement with earlier studies, minichaperones assist folding of some proteins. However, this effect is observed only under conditions where substantial spontaneous folding is also observed and is indistinguishable from that resulting from addition of the nonchaperone protein alpha-casein. By contrast, the full GroE system efficiently promotes folding of several substrates under conditions where essentially no spontaneous folding is observed. These data argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.

D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine.

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.