Relation between flexibility and intrinsically disorder regions in thermosensitive TRP channels reveal allosteric effects.

How a protein propagates the conformational changes throughout its structure remains largely unknown. In thermosensitive TRP channels, this allosteric communication is triggered by ligand interaction or in response to temperature changes. Because dynamic allostery suggests a dynamic role of disordered regions, in this work we set out to thoroughly evaluate these regions in six thermosensitive TRP channels. Thus, by contrasting the intrinsic flexibility of the transmembrane region as a function of the degree of disorder in those proteins, we discovered several residues that do not show a direct correlation in both parameters. This kind of structural discrepancy revealed residues that are either reported to be dynamic, functionally relevant or are involved in signal propagation and probably part of allosteric networks. These discrepant, potentially dynamic regions are not exclusive of TRP channels, as this same correlation was found in the Kv Shaker channel.

Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding and function.

Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.

The interplay of protein-ligand and water-mediated interactions shape affinity and selectivity in the LAO binding protein.

The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine-arginine-ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l-arginine and l-histidine with nm and µm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein-ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein-ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO-binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X-ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine-protein interactions. Furthermore, crystallographic structures revealed that protein-ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state. DATABASE: Structural data are available in the Protein Data Bank database under the accession numbers 6MLE, 6MLN, 6MLG, 6MKX, 6MLI, 6MLA, 6MKU, 6MKW, 6ML0, 6MLD, 6MLV, 6MLO, 6MLP, 6ML9, 6MLJ.

The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction.

It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and ß-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.

On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium.

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.

Energetic effects of magnesium in the recognition of adenosine nucleotides by the F(1)-ATPase beta subunit.

Nucleotide-induced conformational changes of the catalytic beta subunits play a crucial role in the rotary mechanism of F(1)-ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated beta subunit from thermophilic Bacillus PS3 (Tbeta), the binding of this monomer to Mg(II)-free and Mg(II)-bound adenosine nucleotides was characterized using high-precision isothermal titration calorimetry. The interactions of Mg(II) with free ATP or ADP were also measured calorimetrically. A model that considers simultaneously the interactions of Tbeta with Mg.ATP or with ATP and in which ATP is able to bind two Mg(II) atoms sequentially was used to determine the formation parameters of the Tbeta-Mg.ATP complex from calorimetric data. This analysis yielded significantly different DeltaH(b) and DeltaS(b) values in relation to those obtained using a single-binding site model, while DeltaG(b) was almost unchanged. Published calorimetric data for the titration of Tbeta with Mg.ADP [Perez-Hernandez, G., et al. (2002) Arch. Biochem. Biophys. 408, 177-183] were reanalyzed with the ternary model to determine the corresponding true binding parameters. Interactions of Tbeta with Mg.ATP, ATP, Mg.ADP, or ADP were enthalpically driven. Larger differences in thermodynamic properties were observed between Tbeta-Mg.ATP and Tbeta-ATP complexes than between Tbeta-Mg.ADP and Tbeta-ADP complexes or between Tbeta-Mg.ATP and Tbeta-Mg.ADP complexes. These binding data, in conjunction with those for the association of Mg(II) with free nucleotides, allowed for a determination of the energetic effects of the metal ion on the recognition of adenosine nucleotides by Tbeta [i.e., Tbeta.AT(D)P + Mg(II) right harpoon over left harpoon Tbeta.AT(D)P-Mg]. Because of a more favorable binding enthalpy, Mg(II) is recognized more avidly by the Tbeta.ATP complex, indicating better stereochemical complementarity than in the Tbeta.ADP complex. Furthermore, a structural-energetic analysis suggests that Tbeta adopts a more closed conformation when it is bound to Mg.ATP than to ATP or Mg.ADP, in agreement with recently published NMR data [Yagi, H., et al. (2009) J. Biol. Chem. 284, 2374-2382]. Using published binding data, a similar analysis of Mg(II) energetic effects was performed for the free energy change of F(1) catalytic sites, in the framework of bi- or tri-site binding models.

Multithermal titration calorimetry: a rapid method to determine binding heat capacities.

Herein a new method that allows binding DeltaCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DeltaH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature re-adjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DeltaCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme-chitotriose complex as a model system.