Discovery of a thermophilic protein complex stabilized by topologically interlinked chains

Abstract

A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 degrees C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6 A crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.

Figure 1

Abundance of disulfide-bonded proteins in P. aerophilum, as detected by fluorescent labeling. Whole cell lysate was reacted with iodoacetamide (+) to block free (thiol) cysteines. Following blocking, any disulfide bonds present were cleaved by reduction with TCEP and fluorescently labeled with CPM. When iodoacetamide is omitted (−) all cysteines are labeled. A comparison of corresponding lanes shows that a large fraction of P. aerophilum proteins contain disulfide bonds. E. coli cells serve as a control.

Figure 2

A 2-D diagonal gel electrophoresis method for identifying intermolecular disulfide bonded protein complexes. The first separation (1) is performed under non-reducing conditions so that disulfide bonds remain intact. Disulfide bonds are cleaved by reduction with DTT prior to the second electrophoretic separation (2). Proteins involved in intermolecular disulfide bonds appear as spots below the prominent diagonal, while spots above the diagonal mark certain intramolecularly disulfide-bonded proteins whose mobilities are retarded by reduction. Numerous disulfide bonded protein-protein complexes are visible in a cell lysate from (A) P. aerophilum, but not in (B) E. coli used as a control. P. aerophilum protein spots identified by mass spectrometry are numbered as in Table 2.

Figure 3

Crystal structure of P. aerophilum citrate synthase (PaCS). (A) The PaCS homodimer illustrated with the individual subunits colored red and blue. The arrangement of domains is illustrated for the (B) PaCS dimer, (C) individual subunit, and (D) PaCS dimer rotated 90° from B. The N-terminal β-sheet domain is colored yellow, the large domain (helices C-M and S) is colored green, the small domain (helices N-R) is colored red, and the C-terminal domain is colored blue. Disulfide-bonded cysteins are illustrated as spheres.

Figure 4

A multiple sequence alignment of citrate synthase homologues from four thermophilic organisms, illustrating the unusual N-terminus in P. aerophilum. Overall conservation in the remainder of the protein is highlighted with α-helices shaded in grey and β-strand segments boxed. Disulfide-bonded cysteines in P. aerophilum are indicated by asterisks (*).

Figure 5

Intramolecular disulfide bonds leading to topological linkage or catenation of protein chains in P.aerophilum citrate synthase (PaCS). (A) Cartoon representation of interlinked PaCS chains. On the left, the protein backbone of the PaCS dimer is shown in a smoothed form to help clarify the chain topology. The region of the N-terminus (residues 1–24) that differs structurally in comparison to mesophilic homologs is indicated in darker striping. The topological connectivity is illustrated on the right. (B) Close up view of the disulfide bonds between Cys19 and Cys394 for both chains. Electron density maps calculated from diffraction data (based on phases from an omit-model) are shown in blue wireframe. (C) SDS-PAGE gel of purified recombinant PaCS. Under non-reducing (‘Oxd’) conditions, PaCS migrates at a molecular weight consistent with the dimeric form. Following treatment with the chemical reductant TCEP, the reduced (‘Red’) PaCS migrates as a monomer. A minor doublet in the oxidized lane likely corresponds to a mixture of linear and cyclized forms of the monomer. Fluorescent labelling with CPM indicates that no free thiols are present in the oxidized, dimeric form of PaCS.

Figure 6

Thermal denaturation of native and mutant forms of P. aerophilum citrate synthase in 4.5M Gdn-HCl, monitored by circular dichroism. Experiments in the oxidized (thin curve) and reduced forms (thick curve) illustrate the stabilizing contribution from the linkage of the two protein chains.