The genomics of disulfide bonding and protein stabilization in thermophiles

Abstract

Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.

Figure 1. Predicted Protein Disulfide Abundance Across Thermophilic and Mesophilic Microorganisms

For each genome, a colored row illustrates the tendency for cysteine residues in the proteins of that organism to occur close in three-dimensional space to each of the 20 amino acids. The amino acid types are given by their one-letter codes. The values reported are log (base 10) odds ratios, i.e., log ratio of observed over expected occurrences of proximal amino acids, with larger numbers implying a more frequent occurrence of amino acids in proximity. The figure illustrates only a subset of the sequenced organisms analyzed, but includes all the archaeal and bacterial thermophiles. The archaeal and bacterial major branches are noted and species names are provided. Some notable genomes with significant cysteine–cysteine proximity predictions include P. aerophilum, A. pernix, and Py. furiosus. Notably, cysteine–cysteine proximity stands out in thermophiles, particularly in the archaea, when compared with mesophiles such as E. coli. Furthermore, a red asterisk next to an organism name refers to the presence of the PDO protein (see text). Note that branch lengths are based on the National Center for Biotechnology Information taxonomy scheme and are not representative of phylogenetic distance, being used as a helpful visualization tool alone. An extended version featuring all genomes analyzed is available in the supporting online material (Figure S1). A dagger indicates that the value for A. pernix (1.236) exceeds the upper limit (1.0) of the coloring scheme used here.

Figure 2. Correspondence of Growth Temperature and Disulfide Richness

A plot of log ratios of cysteine–cysteine proximity versus optimal growth temperature for 99 sequenced genomes is presented. Optimal growth temperatures were taken from the German Collection of Microorganisms and Cell Cultures (DSMZ) and from genome sequence literature. Organisms are classified by color and symbol shape according to the following scheme: mesophiles that include annotations in the literature suggesting some extremophilic property other than thermophilicity (blue); mesophiles that do not include literature annotations suggesting extremophilic qualities (grey); sulfur-reducing bacteria and archaea (yellow); methanogenic bacteria and archaea (green); non-methanogenic/non-sulfur-reducing thermophiles (red); genomes that contain a PDO protein (triangle) (see text); and genomes that do not contain a PDO protein (circle). The genomes containing the PDO protein fit perfectly into the top right segment of the plot, as illustrated by the box drawn in dotted red lines. Numbers indicate the following organisms: 1, A. pernix; 2, P. aerophilum; 3, S. solfataricus; 4, Py. horikoshii; 5, Py. furiosus; 6, Py. abyssi; 7, S. tokodaii; 8, Thermoplasma volcanium; 9, Thermus thermophilus (both HB8 and HB27); 10, Thermococcus kodakaraensis; 11, T. acidophilum; 12, Aquifex aeolicus; 13, Picrophilus torridus; 14, Thermoanaerobacter tengcongensis; 15, Thermotoga maritima; 16, Symbiobacterium thermophilum; 17, Methanothermobacter thermoautotrophicus; 18, Archaeoglobus fulgidus; 19, Methanococcus jannaschii; 20, Synechococcus elongatus; 21, Geobacillus kaustophilus; 22, Thermosynechococcus elongatus; and 23, Methanopyrus kandleri.

Figure 3. Identification of a Protein Exclusive to Disulfide-Rich Thermophiles

Proteins were searched to find those exclusively present in organisms with high predicted abundance of protein disulfide bonds. Phylogenetic profiles are shown for the seven best protein matches according to our search criteria (see text). All thermophilic genomes are shown across the top, colored according to their predicted disulfide richness (see Figure 1). For each protein row (identified by its GI number), a black box indicates that the homologous protein is present in the genome represented in that column. A single protein, PDO (first profile, GI 18313293, previously annotated as a “glutaredoxin-like protein”, labeled here additionally as “PDO”), is singled out as being most closely correlated with disulfide richness and thermophilicity. Annotation here is taken directly from the annotation provided with the genome. PDO was previously annotated as a “glutaredoxin-like protein” based on its C-terminal similarity to glutaredoxin. A dagger indicates that the value for A. pernix (1.236) exceeds the upper limit (1.0) of the coloring scheme used here.

Figure 4. The PDO Family of Proteins

A multiple sequence alignment of 16 members of the PDO family is shown. The two CxxC motifs are indicated by pairs of red arrows. Each protein is labeled with its organism of origin and GI number. Note the disrupted N-terminal CxxC motif in the P. aerophilum PDO sequence.

Figure 5. The Previously Determined PDO Structure and Evidence that the P. aerophilum Protein Has Similar Disulfide Bonding

(A) The crystal structure of the PDO protein from Py. furiosus [12], PDB accession code 1A8L, showed that it contains two fused thioredoxin-like domains (colored grey and blue), with a single contiguous beta-sheet through both thioredoxin domains such that they effectively form one large domain. Each thioredoxin sub-domain bears a CxxC sequence motif with each pair of cysteines forming a disulfide bond (yellow), consistent with the prediction from the profile analysis that it could play a key role in intracellular protein disulfide-bond formation. Figure 5A was generated using PyMOL [54]. (B) Cysteines in purified recombinant PaPDO exist predominantly in disulfide-bonded form. (Left gel) Denatured PaPDO protein was reacted with the thiol-reactive reagent CPM to fluorescently label cysteines in the presence (+) or absence (−) of the strong reducing agent TCEP. Samples separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis clearly show a minimal labeling of the native protein (in the absence of reductant). (Right gel) Following fluorescence analysis, protein bands were stained with Coomassie Brilliant Blue to determine total protein present. The gel shows that reduced (+) and non-reduced (−) samples contained similar amounts of protein. The slightly lower position of the non-reduced PaPDO compared to reduced PaPDO is attributed to the presence of disulfide bonds in the non-reduced sample, which place constraints on the denatured state of the polypeptide and thus lead to a faster migration rate through the gel.

Figure 6. A Novel P. aerophilum Protein

(A) A ribbon representation of the crystal structure of a novel P. aerophilum protein (GI 18312142) is presented. The six cysteine residues form three disulfide bonds (shown as stick models in yellow). (B–D) Simulated annealing electron density (“omit”) maps in the regions of the disulfide bonds between (B) Cys22 and Cys34, (C) Cys24 and Cys54, and (D) Cys80 and Cys83 are shown. All densities are contoured at 1 standard deviation. Images were generated using PyMOL [54].