The backbone structure of the thermophilic Thermoanaerobacter tengcongensis ribose binding protein is essentially identical to its mesophilic E. coli homolog
© Cuneo et al; licensee BioMed Central Ltd. 2008
Received: 08 November 2007
Accepted: 28 March 2008
Published: 28 March 2008
Comparison of experimentally determined mesophilic and thermophilic homologous protein structures is an important tool for understanding the mechanisms that contribute to thermal stability. Of particular interest are pairs of homologous structures that are structurally very similar, but differ significantly in thermal stability.
We report the X-ray crystal structure of a Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) determined to 1.9 Å resolution. We find that tteRBP is significantly more stable ( app T m value ~102°C) than the mesophilic Escherichia coli ribose binding protein (ecRBP) ( app T m value ~56°C). The tteRBP has essentially the identical backbone conformation (0.41 Å RMSD of 235/271 Cα positions and 0.65 Å RMSD of 270/271 Cα positions) as ecRBP. Classification of the amino acid substitutions as a function of structure therefore allows the identification of amino acids which potentially contribute to the observed thermal stability of tteRBP in the absence of large structural heterogeneities.
The near identity of backbone structures of this pair of proteins entails that the significant differences in their thermal stabilities are encoded exclusively by the identity of the amino acid side-chains. Furthermore, the degree of sequence divergence is strongly correlated with structure; with a high degree of conservation in the core progressing to increased diversity in the boundary and surface regions. Different factors that may possibly contribute to thermal stability appear to be differentially encoded in each of these regions of the protein. The tteRBP/ecRBP pair therefore offers an opportunity to dissect contributions to thermal stability by side-chains alone in the absence of large structural differences.
The mechanisms that contribute to protein thermal stability are varied, subtle, and complex [1–5]. Various contributing factors to thermal stability have been proposed by comparative analysis of thermophilic and mesophilic proteins [4, 6]. Proposed mechanisms can be categorized  generally as contributions by the main-chain structure (new folds , loop shortening ), or by side-chain interactions (increased packing in core  or surface , alteration of amino acid composition [11–13]), post-translational modifications  or co-factor binding [4, 15]). Usually increased stability arises from a combination of sequence- and structure-based adaptations resulting in a collection of improvements in the thermophilic protein compared to its mesophilic counterpart [4, 6, 16, 17]. Consequently, the determination of rules for thermal adaptations are difficult to dissect . Of particular interest, therefore, are pairs of naturally evolved proteins that are structurally very similar but differ substantially in thermal stability. Such pairs allow for the dissection of contributions by amino acid diversity to thermal stability in the absence of structural heterogeneity [17–20]. The structure of the Thermoanaerobacter tengcongensis ribose-binding (tteRBP) presented here reveals that this protein and its counterpart in the mesophilic Escherichia coli (ecRBP) form such a pair.
The ribose-binding proteins are members of the periplasmic binding protein (PBP) superfamily whose members play roles in prokaryotic ABC transport , chemotaxis [22, 23], and intercellular communication  systems. The PBP fold consists of two domains each of which adopts a three-layered α/β/α sandwich motif . The two domains are linked by two or three β-strands that form a flexible hinge which permits the domains of the protein to bend towards each other in response to ligand binding at the interface between the two domains [26–28].
Here we report the high-resolution X-ray crystallographic structure of a ribose binding protein (tteRBP) from the hyperthermophilic bacterium T. tengcongensis (optimal growth temp ~80°C) . We find that tteRBP has high sequence and structural similarity to the mesophilic E. coli RBP (ecRBP), although they differ markedly in their thermal stability. The near identity of backbone structure offers an opportunity to address local encoding of thermal stability by amino acid substitutions.
Results and Discussion
Thermal Stability and Ligand Binding
Binding of ribose to tteRBP was confirmed by observing ligand-mediated changes in the app T m value in the presence of 4.0 M GdCl. Under these conditions in the absence of ribose, the app T m value is 74°C; and 92°C in the presence of 1 mM ribose (Figure 2). The app T m value of the ribose complex in the absence of GdCl is 114°C; the app T m value for ecRBP under equivalent conditions is 72°C (Figure 2).
Overall Structure of tteRBP
Data collection and refinement statistics
Num. of Reflections (working set/test set)
Number of atoms
Bond lengths (Å)
Bond angles (°)
Average B-factor (Å 2 )
Ramachandran outliers (%)
Ramachandran favored (%)
Rotamer outliers (%)
Structural Diversity of tteRBP and ecRBP
Changes in the χ1 values of ecRBP and tteRBP. Non-conservative substitutions (as defined in ; charge inversions are scored as non-conservative here) are underlined.
Hydrogen bonding interactions in tteRBP and ecRBP
Side chain/Side chain
Side chain/Main chain
Main chain/Main chain
Amino Acid Diversity Among tteRBP and ecRBP
Divergence patterns in tteRBP and ecRBP. Classification of amino acids into core, boundary, or surface  allows identification of the regions which are conserved among tteRBP and ecRBP.
Amino acid sequence divergence as a function of core, boundary or surface in tteRBP and ecRBP. Differences are classified as substitutions which are found in tteRBP relative to ecRBP.
Non-Branched to Branched
Branched to Non-Branched
V to I
I to V
We have cloned, expressed, purified, and characterized the structure and stability of the ribose binding protein from the extremophilic bacterium T. tengcongensis. tteRBP is considerably more stable than ecRBP (46°C difference in app T m values of the apo proteins). The amino acid backbone structure of these two proteins are essentially identical (0.41 Å RMSD of 235/271 Cα positions and 0.65 Å RMSD of 270/271 Cα positions), suggesting that all the interactions contributing to differences in thermal stability are encoded entirely in the identity, location, and conformation of the amino acid side-chains.
Comparison of mesophilic and thermophilic protein structures has identified many structural adaptations which are postulated to confer thermal stability [2, 6, 11, 16–18, 38]. Numerous side-chain dependent contributions to thermal stability have been proposed, based on amino acid composition of thermophilic proteins and comparison of mesophilic and thermophilic protein sequences and structures, including; increased number of salt-bridges , differences in polar/apolar exposed and buried surface areas [8, 12, 39], introduction of prolines , introduction of disulfide bridges [41, 42], aromatic interactions , helix dipole stabilization , post-translational modification , alteration of amino acid packing [9, 10, 44] and secondary structure propensity of amino acids [8, 45].
The high structural similarity of the tteRBP/ecRBP pair allows for the dissection of amino acid diversity contributions to thermal stability in the absence of structural heterogeneity. The comparative analysis presented here shows that the substitutions responsible for conferring thermal stability on tteRBP are encoded in side-chain identity and location (core, boundary or surface) which serves to alter surface polarity/charge, removal of unsatisfied core hydrogen bonds and increase in core/boundary side-chain hydrophobicity. In the core of tteRBP there is a bias for the loss of polar amino acids and for the introduction of valine to isoleucine mutations which possibly lower the entropic contribution to the free energy of folding and limits burying core amino acids whose hydrogen bonding potential may remain unsatisfied [38, 46]. The large number of valine to isoleucine substitutions in the tteRBP core and boundary leads to an increase in side-chain hydrophobicity and increased packing [44, 47]. It is additionally observed in the boundary the substitution of non-β-branched amino acids for β-branched residues which has also been postulated to be important in increasing the packing . Additionally, in a trend that is also observed in other thermophilic proteins, the surface of tteRBP is generally more polar and charged with the introduction of an additional three polar residues and eleven charged residues.
The acquisition of thermal stability in tteRBP arises from contributions by side-chain mediated effects alone. This pair of proteins therefore provides a good test case to examine such contributions experimentally and address some long-standing questions in the acquisition of protein stability [1, 5, 49]: where in sequence and structure is stability encoded; how many mutations are needed; are mutations punctuated (single mutants cause large changes) or gradual, independent or correlated? Recent advances in protein fabrication automation  will assist in addressing these questions by enabling rapid construction of the many sequence variants needed.
Cloning Over-expression and Purification
The tte0206 gene was amplified from T. tengcongensis genomic DNA by the sticky-end PCR method using the following primers: PO4-TATGA AAACTATAGG ATTAGTGATATCTACTCTTAACAATCC, and TATGAAAACTATAGG ATTAGTGATATCTACTCTTAACAATCC for the 5' end of the gene; PO4- AATTCTAATGGTGATGGTGATGGTGTGATCCCTGTACATTTTCTTTTGTTATGAGTTTAAGTTCTGC, and CTAATGGTGATGGTGATGGTGTGATCCCTGTACATTTTCTTTTGTTATGAGTTTAAGTTCTGC for the 3' end of the gene . The resulting fragment was cloned into the Nde I/Eco RI sites of a pET21a (Novagen) plasmid for over-expression in E. coli. This ORF lacks the putative periplasmic signal sequence . The coding sequence starting at lysine 40 was cloned in-frame with an ATG start codon. A hexahistidine affinity tag and a glycine-serine linker was fused in-frame at the carboxy terminus to facilitate purification by immobilized metal affinity chromatography (IMAC). Protein concentration was determined spectrophotometrically (ε280 = 3800 M-1cm-1) . The resulting gene product was expressed and purified by IMAC as described . Pooled IMAC fractions were concentrated to 12 mL and were loaded onto a Superdex 26/60 S75 (Amersham) gel filtration column that was previously calibrated with blue dextran, bovine serum albumin, chicken serum albumin, chymotrypsin and lysozyme. tteRBP eluted from the column beginning at the void volume and ending at a calculated hydrodynamic radius corresponding to ~20 KDa. For crystallization and characterization, 10 mL fractions corresponding to a calculated hydrodynamic radius corresponding to an apparent molecular weight of 30 KDa ± 15 kDa, were collected and concentrated to 0.5 mM and dialyzed in 10 mM Tris pH7.8, 20 mM NaCl. An average of 30 mg of pure protein produced per liter of medium.
Circular dichroism (CD) measurements were determined on an Aviv Model 202 circular dichroism spectrophotometer. Thermal denaturations were determined by measuring the CD signal at 222 nm (1 cm path length) as a function of temperature, using 1 μM protein (10 mM Tris-HCl pH7.8, 150 mM NaCl), GdCl at various concentrations, in the presence or absence of 1 mM ribose. Protein samples were incubated for 15 minutes prior to collecting data. Each measurement includes a 3-second averaging time for data collection and a 60 second equilibration period at each temperature. Data was fit to a two-state model which accounts for the native and denatured baseline slopes, to determine the apparent T m values [31, 32]. It is not known whether equilibrium was achieved under these conditions; denaturation midpoint temperatures are therefore reported as apparent values ( app T m ). The app T m values in the absence of denaturant were determined by linear extrapolation .
Crystallization and Data Collection
Ribose was added to tteRBP in 3-fold stoichiometric excess prior to crystallization. tteRBP crystals were grown by micro-batch under paraffin oil in drops that contained 2 μl of the protein solution (0.5 mM) mixed with 2 μl of 0.1 M sodium citrate pH 4.0, 50% (w/v) PEG 1000 and 0.1 M potassium phosphate monobasic. The tteRBP crystals diffract to 1.9 Å resolution, belong to the C2 space group (a = 123.18 Å, b = 35.8 Å, c = 118.03 Å, β = 107.02) and typically grew within three weeks at 17°C (Table 1). No stabilizing cryoprotectant was used and crystals were frozen directly in precipitant solution, mounted in a nylon loop and flash frozen in liquid nitrogen. All data were collected at 100 K at the SER-CAT 22 BM beam line at the Advanced Photon Source. The diffraction data were scaled and indexed using SCALA and XDS [53, 54].
Structure Determination Methods, Model Building and Refinement
The tteRBP structure was determined by molecular replacement using the ribose-bound form of the ribose binding protein from E. coli  as the search model . Rotation, translation, and fitting functions revealed a clear solution yielding higher correlation coefficients and a lower R factor than all the others. Manual model building was carried out in the programs O and COOT and refined using REFMAC5 [55–57]. The final model for the tteRBP complex includes two intact tteRBP monomers (residues 2–275), two ribose molecules, and 346 water molecules. The model exhibits good stereochemistry as determined by PROCHECK and MolProbity; final refinement statistics are listed in Table 1[58, 59]. PDB coordinates and structure factors have been deposited in the RCSB Protein Data Bank under the accession code 2IOY.
This study was funded grant by a grant from HSARPA (W81XWH-05-C-0161) to HWH, a Pioneer Award from the NIH (5 DP1 OD000122-02) to HWH, and a NIH sponsored Biological Chemistry training grant to MJC. The authors would like to acknowledge G. Shirman for protein expression and purification. Data were collected at the Southeast Regional Collaborative Access Team 22-BM at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.
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