Omp85 from the Thermophilic Cyanobacterium Thermosynechococcus elongatus Differs from Proteobacterial Omp85 in Structure and Domain Composition

Thomas Arnold,Kornelius Zeth,Dirk Linke

doi:10.1074/jbc.m110.112516

Abstract

Omp85 proteins are essential proteins located in the bacterial outer membrane. They are involved in outer membrane biogenesis and assist outer membrane protein insertion and folding by an unknown mechanism. Homologous proteins exist in eukaryotes, where they mediate outer membrane assembly in organelles of endosymbiotic origin, the mitochondria and chloroplasts. We set out to explore the homologous relationship between cyanobacteria and chloroplasts, studying the Omp85 protein from the thermophilic cyanobacterium Thermosynechococcus elongatus. Using state-of-the art sequence analysis and clustering methods, we show how this protein is more closely related to its chloroplast homologue Toc75 than to proteobacterial Omp85, a finding supported by single channel conductance measurements. We have solved the structure of the periplasmic part of the protein to 1.97 Å resolution, and we demonstrate that in contrast to Omp85 from Escherichia coli the protein has only three, not five, polypeptide transport-associated (POTRA) domains, which recognize substrates and generally interact with other proteins in bigger complexes. We model how these POTRA domains are attached to the outer membrane, based on the relationship of Omp85 to two-partner secretion system proteins, which we show and analyze. Finally, we discuss how Omp85 proteins with different numbers of POTRA domains evolved, and evolve to this day, to accomplish an increasing number of interactions with substrates and helper proteins.

Highlights

Referred to as BamA, form a protein complex with three to four lipoproteins (BamB–E) designated the Bam complex in Escherichia coli [3, 4]
This homology and the conserved function of the complexes in prokaryotes and in eukaryotic organelles are major molecular evidence for the endosymbiont theory, which states that chloroplasts arose from cyanobacteria, and mitochondria from primitive ␣-proteobacteria, which were incorporated into the first simple eukaryotic cells [11,12,13,14]
Sam50 is the only member of the Omp85 family in mitochondria involved in the assembly and insertion of outer membrane proteins, there exist at least two distinct proteins in the outer envelope of chloroplasts, Toc75-III and Toc75-V (Oep80 in Arabidopsis), and several isoforms thereof [15, 16]

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—All constructs were obtained by PCR amplification using genomic DNA isolated from T. elongatus BP1 cells. Purification of TeOmp and TeOmp85-C—The cells were resuspended in 30 ml of resuspension buffer (150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 20 mM Tris-HCl, pH 8.5, and a pinch of DNase I) and lysed using a French press. The pelleted inclusion bodies were resuspended in 150 mM NaCl, 20 mM Tris-HCl, pH 8.5. For fast protein liquid chromatography purification, an aliquot of inclusion body suspension was solubilized in 6 M guanidinium HCl, 500 mM NaCl, 10% glycerol, 20 mM Tris-HCl, pH 8.5, and applied on a nickel-nitrilotriacetic acid column (nickel-Sepharose, GE Healthcare) equilibrated in the same buffer. Purification of TeOmp85-N—The cells were resuspended in 30 ml of resuspension buffer (150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 20 mM Tris-HCl, pH 8.5, and a pinch of DNase I) and lysed using a French pressure cell. Data collectiona Wavelength Space group Resolution Cell constants Unique reflections Redundancy Completeness Rmerge-F I/␴(I) Wilson B-factor

Phasing statistics

Fusobacterium nucleatum

RESULTS

BamA and Neisseria meningitidis

POTRA domains in proteobacterial

DISCUSSION