Novel molecular architecture of the multimeric archaeal PEP-synthase homologue (MAPS) from Staphylothermus marinus

The phosphoenolpyruvate (PEP)-synthases belong to the family of structurall y and functionally related PEP-utilizing enzymes. The only archaeal member of this family characterized thus far is the Multimeric Archaeal PEP-Syntha se homologue from Staphylothermus marinus (MAPS). This protein complex diff ers from the bacterial and eukaryotic representatives characterized to date in its homomultimeric, as opposed to dimeric or tetrameric, structure. We have probed the molecular architecture of MAPS using limited proteolytic di gestion in conjunction with electron microscopic, biochemical, and biophysi cal techniques. The 2.2 MDa particle was found to be organized! in a concen tric fashion. The 93.7 kDa monomers possess a pronounced tripartite domain structure and are arranged such that the N-terminal domains form an outer s hell, the intermediate domains form an inner shell, and the C-terminal doma ins form a core structure responsible for the assembly into a multimeric co mplex. The core domain was shown to be capable of assembling into the nativ e multimer by recombinant expression in Escherichia coli. Deletion mutants as well as a synthetic peptide were investigated for their state of oligome rization using native polyacrylamide gel electrophoresis, molecular sieve c hromatography, analytical ultracentrifugation, circular dichroism (CD) spec troscopy, and chemical cross-linking. Our data confirmed the existence of a short C-terminal, tx-helical oligomerization motif that had been suggested by multiple sequence alignments and secondary structure predictions. We pr opose that this motif bundles the monomers into six groups of four. An addi tional formation of 12 dimers between globular domains from different bundl es leads to the multimeric assembly. According to our model, each of the si x bundles of globular domains is positioned at the corners of an imaginary octahedron, and the helical C-terminal segments are oriented towards the ce ntre of the particle. The edges of the octahedron represent the dimeric con tacts. Phylogenetic analysis suggests that the ancient predecessor of this family of enzymes contained the C-terminal oligomerization motif as a featu re that was preserved in some hyperthermophiles. (C) 1999 Academic Press.