The extracellular subunit interface of the 5-HT3 receptors: a computational alanine scanning mutagenesis study

The functional serotonin 5-HT type-3 (5-HT(3)) receptor, the target of many neuroactive drugs, is known to be a pseudo-symmetric pentamer made either of five identical subunits A (homomeric 5-HT(3A)-R) or of subunits A and B (heteromeric 5-HT(3A/B)-R) in a still debated arrangement. The serotonin binding site is located in the extracellular region, at the interface between two monomers, called the principal and the complementary subunits. The results of molecular dynamics simulations and computational alanine scanning mutagenesis studies applied here to the homomeric human 5-HT(3A)-R disclose an aromatic "hot" cluster in the centre of the interface formed by residues W178 (principal subunit), Y68, Y83, W85 and Y148 (complementary subunit). Moreover, investigation of the coupling of agonist/antagonist binding to channel activation/inactivation points out the presence of two putative functional pathways at the subunit interface: W116-H180-L179-W178-E124-F125 (principal subunit) and Y136-Y138-Y148-W85-(P150) (complementary subunit), where W178 and Y148 appear to be critical residues for the binding/activation mechanism. Finally, direct comparison of the main features shown by the AA interface in the human 5-HT(3A)-R with those of the BB interface in the homopentameric human 5-HT(3B)-R provides interesting clues about the possible reasons that cause the 5-HT(3B)-R not to be functional.     

The 5-HT3AB Receptor Shows an A3B2 Stoichiometry at the Plasma Membrane

The 5-HT₃AB receptor is the best-characterized heteropentameric 5-HT₃ receptor. Under conditions of heterologous expression, the 5-HT₃AB receptor shows a single functionally resolvable population, suggesting the presence of a unique subunit stoichiometry; however, conflicting previous reports have suggested two different possible stoichiometries. Here we isolate plasma membrane sheets containing assembled receptors from individual HEK293T cells. We then determine the stoichiometry of 5-HT₃AB receptors on the plasma membrane by fluorescence methods, employing meCFP- and meYFP-labeled A and B subunits. Over a wide range of cDNA transfection ratios, fluorescence intensity ratios are closest to values that correspond to a subunit ratio of A₃B₂. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3–6%) to the subunit ratios and provide independent support for a predominantly A₃B₂ stoichiometry on the plasma membrane sheets. Twin FRET efficiencies support these data, also suggesting that the two B subunits are nonadjacent in most of the heteropentamers. The high-frequency variant HTR3B p.Y129S (c.386A>C, rs11767445), linked to psychiatric disease, also forms A₃B₂ receptors on the plasma membrane. The 5-HT₃B Y129S, subunit incorporates in a slightly (11–14%) more efficient manner than the common variant. In general, most of the subunits reside within the cell. In contrast to the findings for the plasma membrane, the relative abundances and FRET characteristics of intracellular subunits depend strongly on the transfection ratio. The straightforward and unambiguous combination of plasma membrane-sheet isolation, fluorescence intensity ratios, and FRET is a generally promising procedure for determining stoichiometry of proteins on the plasma membrane.     

The 5-HT3AB Receptor Shows an A3B2 Stoichiometry at the Plasma Membrane

The 5-HT₃AB receptor is the best-characterized heteropentameric 5-HT₃ receptor. Under conditions of heterologous expression, the 5-HT₃AB receptor shows a single functionally resolvable population, suggesting the presence of a unique subunit stoichiometry; however, conflicting previous reports have suggested two different possible stoichiometries. Here we isolate plasma membrane sheets containing assembled receptors from individual HEK293T cells. We then determine the stoichiometry of 5-HT₃AB receptors on the plasma membrane by fluorescence methods, employing meCFP- and meYFP-labeled A and B subunits. Over a wide range of cDNA transfection ratios, fluorescence intensity ratios are closest to values that correspond to a subunit ratio of A₃B₂. Förster resonance energy transfer (family FRET) efficiencies provide minor corrections (3–6%) to the subunit ratios and provide independent support for a predominantly A₃B₂ stoichiometry on the plasma membrane sheets. Twin FRET efficiencies support these data, also suggesting that the two B subunits are nonadjacent in most of the heteropentamers. The high-frequency variant HTR3B p.Y129S (c.386A>C, rs11767445), linked to psychiatric disease, also forms A₃B₂ receptors on the plasma membrane. The 5-HT₃B Y129S, subunit incorporates in a slightly (11–14%) more efficient manner than the common variant. In general, most of the subunits reside within the cell. In contrast to the findings for the plasma membrane, the relative abundances and FRET characteristics of intracellular subunits depend strongly on the transfection ratio. The straightforward and unambiguous combination of plasma membrane-sheet isolation, fluorescence intensity ratios, and FRET is a generally promising procedure for determining stoichiometry of proteins on the plasma membrane.     

Phosphatidylinositol 4-phosphate 5-kinase Iγ_v6, a new splice variant found in rodents and humans

It has been shown that anti-cancer drug induces secretion of serotonin (5-HT) from small intestine which activates serotonin type 3 (5-HT₃) receptor to cause nausea and vomiting. In general, antagonist for 5-HT₃ receptor is used as anti-emetics during chemotherapy. However, we found that anti-cancer drug irinotecan itself inhibits 5-HT-gated current through the homomeric 5-HT_3A and heteromeric 5-HT_3AB receptor in a concentration-dependent manner. The inhibitory effect of irinotecan on 5-HT_3A receptor was more potent than that on 5-HT_3AB receptor. On the other hand, SN-38, a metabolite of irinotecan, had no effect on the responsiveness. Our findings suggest that irinotecan itself could have anti-emetic activities through inhibition of the 5-HT_3A and 5-HT_3AB receptor.     

Human α1-adrenoceptor subtype selectivity of substituted homobivalent 4-aminoquinolines

A series of ring-substituted ethyl- and heptyl-linked 4-aminoquinoline dimers were synthesized and evaluated for their affinities at the 3 human α₁-adrenoceptor (α₁-AR) subtypes and the human serotonin 5-HT_1A-receptor (5-HT_1A-R). We find that the structure-specificity profiles are different for the two series at the α₁-AR subtypes, which suggests that homobivalent 4-aminoquinolines can be developed with α₁-AR subtype selectivity. The 8-methyl (8-Me) ethyl-linked analogue has the highest affinity for the α_1A-AR, 7 nM, and the greatest capacity for discriminating between α_1A-AR and α_1B-AR (6-fold), α_1D-AR (68-fold), and the 5-HT_1A-R (168-fold). α_1B-AR selectivity was observed with the 6-methyl (6-Me) derivative of the ethyl- and heptyl-linked 4-aminoquinoline dimers and the 7-methoxy (7-OMe) derivative of the heptyl-linked analogue. These substitutions result in 4- to 80-fold selectivity for α_1B-AR over α_1A-AR, α_1D-AR, and 5-HT_1A-R. In contrast, 4-aminoquinoline dimers with selectivity for α_1D-AR are more elusive, since none studied to date has greater affinity for the α_1D-AR over the other two α₁-ARs. The selectivity of the 8-Me ethyl-linked 4-aminoquinoline dimer for the α_1A-AR, and 6-Me ethyl-linked, and the 6-Me and 7-OMe heptyl-linked 4-aminoquinoline dimers for the α_1B-AR, makes them promising leads for drug development of α_1A-AR or α_1B-AR subtype selective ligands with reduced 5-HT_1A-R affinity.     

Model studies on a synthetically facile series of N-substituted phenyl-N′-pyridin-3-yl ureas leading to 1-(3-pyridylcarbamoyl) indolines that are potent and selective 5-HT2C/2B receptor antagonists

A model series of 5-HT_2C antagonists have been prepared by rapid parallel synthesis. These N-substituted phenyl-N′-pyridin-3-yl ureas were found to have a range of 5-HT_2C receptor affinities and selectivities over the closely related 5-HT_2A receptor. Extrapolation of simple SAR, derived from this set of compounds, to the more active but synthetically more complex 1-(3-pyridyl-carbamoyl)indoline series allowed us to target optimal substitution patterns and identify potent and selective 5-HT_2C/2B antagonists.     

Synthesis and structure–activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists. Part 2

We previously reported that the novel dual 5-HT_2B and 5-HT₇ receptor antagonist N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (4) exerted a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs. To develop a synthetic strategy, we performed docking studies of lead compound 4 bound to 5-HT_2B and 5-HT₇ receptors, and observed that the carbonyl guanidine group forms a tight interaction network with an active center Asp (D135:5-HT_2B, D162:5-HT₇), Tyr (Y370:5-HT_2B, Y374:5-HT₇) and aromatic residue (W131:5-HT_2B, F158:5-HT₇). Based on molecular modeling results, we optimized the substituents at the 5- to 8-position and 9-position of the fluorene ring and identified N-(diaminomethylene)-9-hydroxy-9-methyl-9H-fluorene-2-carboxamide (24a) exhibits potent affinity for 5-HT_2B (K_i = 4.3 nM) and 5-HT₇ receptor (K_i = 4.3 nM) with high selectivity over 5-HT_2A, 5-HT_2C, α₁, D₂ and M₁ receptors. Compound 24a reversed the hypothermic effect of 5-carboxamidotryptamine (5-CT) in mice and also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered at 30 mg/kg. Compound 24a is therefore a promising candidate for a novel class of anti-migraine agent without any adverse effects.     

Dopamine D1 receptor and serotonin 5-HT1A receptor agonist effects of the natural product (–)-stepholidine: molecular modelling and dynamics simulations

In this study, by homology modelling and molecular dynamics (MD) simulation, models of l-stepholidine (l-SPD) activating the 5-HT_1A and D₁ receptors were constructed. In 100-ns MD simulations, the D₁ and 5-HT_1A receptors were activated by the partial agonist l-SPD, conforming with the global toggle switch activation model and the sequential activation model. The residues Y^7.53 and Y^5.58 swing significantly between different transmembrane (TM) domains after activation. Similarities between D₁ and 5-HT_1A included (1) the outward motion of TM-5; (2) the ionic lock was independent of the tilt of TM-6 and (3) there was an apparent bending of TM-6, and the ring of l-SPD formed strong π–π interactions with residue W^6.48. Differences between the two included the following: (1) in 5-HT_1A, l-SPD formed a hydrogen bond with Ala172^5.46 of TM-5, and the intracellular end of TM-5 moved outward slowly; that hydrogen bond did not form with the D₁ receptor; (2) l-SPD formed stronger interactions with D^3.32 and W^6.48 in the D₁ receptor than in the 5-HT_1A receptor and (3) the hydrogen bonding network was somewhat different in SPD-5-HT_1A and SPD-D₁ receptors. We propose the interaction between l-SPD and D^3.32 or/and W^6.48 is the original driving force during the whole activation process.     

Structural insights into the South African HIV-1 subtype C protease: impact of hinge region dynamics and flap flexibility in drug resistance

The HIV protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. Resistance of HIV protease to protease inhibitors (PIs) is problematic for the effective treatment of HIV infection. The South African HIV-1 subtype C protease (C-SA PR), which contains eight polymorphisms relative to the consensus HIV-1 subtype B protease, was expressed in Escherichia coli, purified, and crystallized. The crystal structure of the C-SA PR was resolved at 2.7?Å, which is the first crystal structure of a HIV-1 subtype C protease that predominates in Africa. Structural analyses of the C-SA PR in comparison to HIV-1 subtype B proteases indicated that polymorphisms at position 36 of the homodimeric HIV-1 protease may impact on the stability of the hinge region of the protease, and hence the dynamics of the flap region. Molecular dynamics simulations showed that the flap region of the C-SA PR displays a wider range of movements over time as compared to the subtype B proteases. Reduced stability in the hinge region resulting from the absent E35-R57 salt bridge in the C-SA PR, most likely contributes to the increased flexibility of the flaps which may be associated with reduced susceptibility to PIs.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:36     

The Serotonin 5-HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin-Mediated Phosphoinositide Hydrolysis

Abstract: Serotonin (5-hydroxytryptamine; 5-HT) 5-HT_2A and 5-HT_2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT_2A-linked and 5-HT_2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca²⁺ (300 nM) and GTPγS (1 µM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT_2A antagonist, spiperone, and the nonselective 5-HT_2A/2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT_2C receptors as opposed to 5-HT_2A receptors. Radioligand binding assays with [³H]RP 62203 and [³H]-mesulergine were used to quantify 5-HT_2A and 5-HT_2C sites, respectively, in caudate. From these data, the B_max for caudate 5-HT_2A sites and 5-HT_2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT_2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT_2A- and 5-HT_2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT_2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT_2C sites. The significance of these observations with respect to the physiological function of 5-HT_2C receptors is discussed.     

Prefrontal Serotonergic Denervation Induces Increase in the Density of 5-HT<Subscript>2A</Subscript> Receptors in Adult Rat Prefrontal Cortex

The 5-HTergic system and particularly 5-HT_2A receptors have been involved in prefrontal cognitive functions, but the underlying mechanisms by which the serotonin (5-HT) system modulates these processes are still unclear. In this work, the effects of prefrontal 5-HTergic denervation on the density and expression levels of 5-HT_2A receptors were evaluated by immunohistochemical and molecular biology studies in the prefrontal cortex (PFC). The [³H]-Ketanserin binding study revealed an increase in the B_max, along with no change in the binding affinity (K_D) for 5-HT_2A receptors. The increase in PFC of 5-HT_2A receptor density in response to denervation was accompanied by increase in 5-HT_2A receptor mRNA and protein levels. This increase in the number of 5-HT_2A receptors may be interpreted as an adaptive plastic change, i.e., hypersensitivity; resulting from the selective pharmacological lesion of the raphe-proceeding 5-HTergic fibers to the PFC. Based on previous evidence, this could be strongly related to the abnormal expression of short-term memory.     

Synthesis and structure-activity relationship of 1H-indole-3-carboxylic acid pyridine-3-ylamides: a novel series of 5-HT2C receptor antagonists

A novel series of 1H-indole-3-carboxylic acid pyridine-3-ylamides were synthesized and identified to show high affinity and selectivity for 5-HT_2C receptor. Among them, 1H-indole-3-carboxylic acid[6-(2-chloro-pyridin-3-yloxy)-pyridin-3-yl]-amide (15k) exhibits the highest affinity (IC₅₀ = 0.5 nM) with an excellent selectivity (>2000 times) over other serotonin (5-HT_1A, 5-HT_2A, and 5-HT₆) and dopamine (D₂–D₄) receptors.     

Up-Regulation of 5-HT2B Receptor Density and Receptor-Mediated Glycogenolysis in Mouse Astrocytes by Long-Term Fluoxetine Administration

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca²⁺ concentration ([Ca²⁺]_i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca²⁺]_i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT₂ receptor that is different from the 5-HT_2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT_2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT_2A and 5-HT_2B receptors, but not for the 5-HT_2C receptor, identifying the 5-HT₂ receptor activated by fluoxetine as the 5-HT_2B receptor, the most recently cloned 5-HT₂ receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [³H]mesulergine, a ligand for all three 5-HT₂ receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT_2B-mediated effects are initially down-regulated and subsequently up-regulated.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.     

Evidence-based docking of the urease activation complex

Abstract

^{1 1}Both authors share senior authorship. Ureases require accessory proteins for their activation and proper function. In Klebsiella aerogenes, UreD, UreF, UreG, and UreE are sequentially complexed to UreABC as required for its activation. Until now, only low-resolution structures are available for this activation complex. To circumvent such limitation, our work intends to provide an atomic-level model for the (UreABC–UreDFG)₃ complex from K. aerogenes, by employing comparative modeling associated to sequential macromolecular dockings, validated through small-angle X-ray scattering profiles and comparison with results from cross-linking, mutagenesis, and pull-down experiments. Additionally, normal mode analyses of the obtained complex supported the characterization of the elevated flexibility of both UreD–UreF dimer and (UreABC–UreDFG)₃ oligomer, explaining the previously observed diffuse binding of UreD to the apoenzyme. The model shown here is the first atomic-level depiction of this complex, a required step for the unraveling of the urease activation process.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:6     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

Modulation of Antagonist Binding to Serotonin1A Receptors from Bovine Hippocampus by Metal Ions

1. The serotonin_1A(5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Although specific 5-HT_1Aagonists have been discovered more than a decade back, the development of selective 5-HT_1Aantagonists has been achieved only recently.2. We have examined the modulation of the specific antagonist [³H]p-MPPF binding to 5-HT_1Areceptors from bovine hippocampal membranes by monovalent and divalent metal ions. Our results show that the antagonist binding to 5-HT_1Areceptors is inhibited by both monovalent and divalent cations in a concentration-dependent manner. This is accompanied by a concomitant reduction in binding affinity.3. Our results also show that the specific antagonist p-MPPF binds to all available receptors in the bovine hippocampal membrane irrespective of their state of G-protein coupling and other serotonergic ligands such as 5-HT and OH-DPAT effectively compete with the specific antagonist [³H]p-MPPF.4. These results are relevant to ongoing analyses of the overall modulation of ligand binding in G-protein-coupled seven transmembrane domain receptors.     

Docking and molecular dynamics studies of peptide inhibitors of ornithine decarboxylase: a rate-limiting enzyme for the metabolism of Fusarium solani

Fusarium solani causes a wide variety of diseases in plants. Polyamine biosynthesis is responsible for the growth and pathogenicity of the fungus. The initial step of this pathway involves the decarboxylation of ornithine to putrescine, and is catalyzed by the enzyme ornithine decarboxylase (ODC). Inhibiting this process may be a promising approach for the management of fungal disease in various crops. Therefore, there is a need to develop inhibitors of ODC that have higher binding capacity than ornithine. Fifteen peptides were designed and modeled based on physicochemical properties of residues in the active site of ODC. The peptide GLIWGNGPF showed the highest dock score. It is assumed that the de novo design of peptides could be a potential approach to inhibit polyamine biosynthesis. Molecular dynamics studies make an important contribution to understanding the effect of the binding of peptides and the stability of an ODC-peptide complex system.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:8.     

Molecular dynamics simulations of the thermal stability of tteRBP and ecRBP

Molecular dynamics simulations were performed for investigating the thermal stability of the extremely thermophilic Thermoanaerobacter tengcongensis ribose binding protein (tteRBP) and the mesophilic homologous Escherichia coli ribose binding protein (ecRBP). The simulations for the two proteins were carried out under the room temperature (300?K) and the optimal activity temperature (tteRBP 375?K and ecRBP 329?K), respectively. The comparative analyses of the trajectories show that the two proteins have stable overall structures at the two temperatures; further analyses indicate that they both have strong side-chain interactions and different backbone flexibilities at the different temperatures. The tteRBP 375?K and ecRBP 329?K have stronger internal motion and higher flexibility than tteRBP 300?K and ecRBP 300?K, respectively, it is noted that the flexibility of tteRBP is much higher than that of ecRBP at the two temperatures. Therefore, tteRBP 375?K can adapt to high temperature due to its higher flexibility of backbone. Combining with the researches by Cuneo et al., it is concluded that the side-chain interactions and flexibility of backbone are both the key factors to maintain thermal stability of the two proteins.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:22