Domain fold
It has been shown that the domain fold play a major role in substrate selection by GroEL in
E. coli and also the analysis of the domain fold distribution according to the SCOP database
revealed a clear impact of the domain fold on the substrate spectra of the group I and the group II chaperonins (Figure 48).
Figure 50 : Representative structure images of domain folds of chaperonin substrates (A) A significant number of substrate proteins of the group I chaperonin exhibit c-class domain folds (mainlyα/β folds). The most abundant are the TIMβ/α barrel fold (c.1;parallel beta-sheet barrel), Adenine nucleotide alpha hydrolase-like (c.26; core : 3 layers, a/b/a; parallel beta sheet) and UDP-Glycosyltransferase/glycogen phosphorylase fold (c.87; 3 layers, a/b/a; parallel beta sheet of 6 strands). (B) Proteins with the ribonuclease H- like motif (c.55; a/b/a; mixed beta-sheet) and the OB-fold domain of mainly beta sheets (class b) show a strong dependency on the group II chaperonin for folding assistance.
In M. mazei several members of the c-class fold, mainlyα/β folds, showed a significantly increased interaction with MmGroEL. Most prevalent among these MmGroEL dependent c-
class folds (mainlyα/β folds) was the TIM β/α barrel (c.1) fold- consistent with the study on the
EcGroEL interaction proteome. Some representatives of this fold that were identified in the
lysate were not found to interact with a chaperonin. This makes it unlikely that the TIMβ/α barrel fold is a sufficient criterion to determine interaction of a protein with MmGroEL. The c.1
fold is classified into a several superfamilies, and the most prominent superfamily found among
MmGroEL-, 2.4% of MmThs substrates, 1% in lysate); only one protein sharing the c.1.2.
superfamily domain fold was identified in the lysate but not found among the MmGroEL
interactors.
All members of the c-class fold adenine nucleotide alpha hydrolase-like (c.26) that were found in the lysate were also identified on MmGroEL, some were also found to interact with the
group II chaperonin. Substrates sharing the c.26 domain fold are mainly represented by two superfamilies: the nucleotidylyl transferase (c.26.1) superfamily and the adenine nucleotide alpha hydrolases-like (c.26.2) superfamily. No correlation of the c.26 fold or its superfamilies with any other structural features (i.e. net charge, hydrophobicity, molecular weight) could be detected that could determine interaction with MmGroEL. Depending on the individual protein,
overlapping substrates sharing the c.26 domain fold show preference for either of the two chaperonins.
The third c-class domain fold, which is frequently found with MmGroEL, is categorized as
the UDP-glycosyltransferase/glycogen phosphorylase fold. This fold is represented by only one superfamily in M. mazei: the UDP-Glycosyltransferase/glycogen phosphorylase (c.87.1)
superfamily. Proteins sharing this domain fold might be of a low abundance in the lysate, because they could only be detected among MmGroEL/substrate complexes and not in the
lysate. Two proteins with this fold domain were also identified on MmThs. These proteins
(gi|0905621 and gi|20906666) were found at lower abundance on MmThs compared to
MmGroEL. Interestingly these proteins are negatively charged (-2.8168 and -0.5864) and have
a low hydrophobicity index (0.4085 and 0.39) compared to strict MmGroEL interactors (0.43 at average), such features are typically found among MmThs (specific) substrates.
The most prominent domain fold found to interact with MmThs exhibits the Ribonuclease H-
like motif (c.55). With the exception of two proteins, all members of this fold class that were identified in the lysate interact with MmThs: (i) the archaeal “conserved protein” (gi|20905336)
was detected only in one of the MmThs substrate samples and was therefore not categorized
as MmThs substrate and (ii) the DNA mismatch repair protein mutS (gi|20906191), which is a
highly conserved protein (evolutionary scope of 0.77) and interacts specifically with MmGroEL.
Interestingly, all but one of the c.55 MmThs substrates are negatively charged, which should
ATPase domain (c.55.1) superfamily (9 of the 13 substrates) and these proteins exclusively interact with the group II chaperonin, consistent with actin being an obligate substrate of the eukaryotic group II chaperonin TRiC. Surprisingly, 10 of the MmThs interacting proteins sharing
the c.55 fold are of bacterial origin, but do not show a high conservation with evolutionary scope values below 0.75 - typically for bacterial proteins that interact with the archaeal chaperonin. Additionally 4 of these 6 bacterial proteins exhibit a size between 62 kDa and 74 kDa that is not favoured by the MmGroEL interactors, but more frequently found among MmThs (specific)
substrates.
MmThs substrates also include members of the b-class domain folds, such as the OB-fold
(b.40). Most proteins of M. mazei exhibiting the b.40 domain fold are found among the
superfamily of nucleic acid-binding proteins (b.40.4) and interact strictly with MmThs, with the
exception of the Lysyl-tRNA synthetase2 (gi|20906448). This protein is highly conserved (evolutionary scope: 0.77) and the only OB fold protein in M. mazei of bacterial origin. Only one
proteinwith the b.40 domain fold that was identified in the lysate, the inorganic pyrophophatase (gi| 20905923), which shows no association with any chaperonin. This has a fold of the superfamily inorganic pyrophophatases (b.40.5). Interestingly, proteins sharing the OB fold are strongly charged, but in contrast to the finding above, that suggests a preferential interaction of
MmThs with negatively charged proteins, 4 of the 10 proteins are positively charged.
Unknown folds
The structural analysis is based on a limited number of the substrate proteins, as structural data about 30% of the total soluble proteome is still missing. Interestingly comparison of the abundance of these unknown folds between the lysate fraction and the substrate sets revealed a strong bias to unknown folds among MmThs specific substrates. Proteins of unknown folds
are found twice as often in the MmThs substrate set as in the lysate or in the MmGroEL
substrate set. This imbalance might have established because of technical problems in protein preparation for crystallisation. For purification, proteins are routinely over-expressed in bacterial cells and thus the overproduction of protein with a strong dependence on group II chaperonins would be doomed to fail in the bacterial host. This problem is less pronounced for MmGroEL dependent proteins, as GroEL/ES is available at least to a limited extent or it might even be co-
overexpressed. In addition, the large scale crystallisation attempts using both E. coli as well as
S. cerevisiae for high through-put purification of proteins might be again limited by the
availability of the group II chaperonin in the yeast system (0.3 µM).
This limited accessibility for proteins that depend on the assistance of a group II chaperonin by heterologous expression might also be a major cause for the substantial fraction of proteins among the MmThs substrates that can not be assigned to any known function. The inability to
produce these proteins efficiently in the bacterial host is a major problem in studying the function of these proteins. Apart from this technically caused correlation, the function of a protein seems to have no direct influence on its chaperonin requirement.