An Unusual Large Protein Complex With Eight-Fold Symmetry is a Major Cellular Component of Thermophilic Archaebacteria
Authors: Phipps Barry M. , Alber Angelika , Stetter Karl O. , Lottspeich Friedrich , Baumeister Wolfgang 
Published: June 2020

Thermophilic archaebacteria produce several large cytoplasmic protein complexes which can be subdivided into recognizable types on the basis of their appearance in the electron microscope. One example is the multicatalytic proteinase (MCP) of Thermoplasma acidophilum, which is related to the mammalian MCP. We have been studying a large ring-shaped protein complex with striking 8-fold symmetry which we have so far identified in two species of Pyrodictium and in Thermoplasma acidophilum (Figs. 1,2). A similar complex appears to exist in Sulfolobus and Archaeoglobus. All of these organisms are thermophilic archaebacteria, with growth temperature optima ranging from 60°C to 105°C, but they nevertheless span a large phylogenetic distance. In Pyrodictium and Thermoplasma the complex constitutes a major fraction of the total cellular protein. Negatively-stained complexes from Pyrodictium brockii adopt 2 different orientations with respect to the carbon film, producing 2 distinct views which we designate “end-on” (ring-shaped) and “side-on” (striated) (Figs. 1, 2). The complexes exhibit some tendency to associate in short chains, especially when in the side-on orientation. The structure was examined by separately selecting end-on and side-on views and subjecting them to single particle averaging via correlation methods using the EM and Semper programme systems. An average of end-on views, shown in Figure 3, reveals 8 well-defined uniformly spaced centres of mass arranged in a ring around a central stain-filled hole. Side-on views were less frequently found and rather more variable in appearance. However a satisfactory average was obtained with particles extracted from several micrographs (Fig. 4). The complex in this orientation has 2-fold rotational symmetry around an axis perpendicular to the plane of the image and passing through the centre of the complex. While the upper and lower halves of the image appear to be mirror symmetric, this cannot be the case for chiral protein molecules. The upper and lower halves each consist of 2 strong masses in the centre and 2 weak masses peripherally. We interpret these observations in terms of a complex composed of 2 stacked disks each comprising 8 roughly ellipsoidal subunits arrayed around a central channel, as shown in Fig, 5. The overall shape of the complex is that of a cylinder 16 nm in diameter and 15.5 nm in height.

The complex was purified from membrane-free French press lysates of Pyrodictium occultum cells by DEAE-Sephacel chromatography, resolution of the eluted protein on sucrose-glycerol and glycerol gradients, and chromatography on a Mono-S column. The purified complex produces 2 bands of approximately equal staining intensity at Mr ca. 60,000 in SDS-PAGE. Based on the dimensions of the particle derived from the above averages, one can estimate the molecular mass to be in the range of 1.1-2.0 × 106. A complex composed of 16 subunits of mass 60,000 would have an Mr of 1.0 × 106, suggesting that the subunits visualized in the model could be monomers or dimers of these polypeptides. The NH2-terminal amino acid sequence of an internal tryptic peptide from one of the polypeptides is: (asp)-(val)-glu-asn-ala-tyr-ile-val-leu-leu-asp-ala-pro-leu-glu-val-glu-lys. It bears no homology to known protein sequences. The purified complex has a moderate ATPase activity at 85°C in the presence of Mg2+. We have not yet been able to assign a function to the complex, but we note that groE and hsp60, the E. coli and mitochondrial chaperonins which appear to catalyze correct protein folding during heat stress and protein assembly and secretion, are both large ring-shaped complexes possessing ATPase activity and are composed of polypeptides of Mr 60.000. Although the symmetry of the archaebacterial complexes is different (groE and hsp70 have 7-fold symmetry), we speculate that they might serve a constitutive chaperonin function in thermophilic archaebacteria. Current studies are aimed at determining the 3D structure of the complex from Pyrodictium, assaying it for chaperonin-like activity, and analysing the sequences of proteolytic peptide fragments to ascertain if homology exists with chaperonins or other known proteins.