Altered Conformational Landscape upon Sensing Guanine Nucleotides in a Disease Mutant of Elongation Factor-like 1 (EFL1) GTPase.

The final maturation step of the 60S ribosomal subunit requires the release of eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) to enter the pool of mature ribosomes capable of engaging in translation. This process is mediated by the concerted action of the Elongation Factor-like 1 (human EFL1, yeast Efl1) GTPase and its effector, the Shwachman-Bodian-Diamond syndrome protein (human SBDS, yeast Sdo1). Mutations in these proteins prevent the release of eIF6 and cause a disease known as Shwachman-Diamond Syndrome (SDS). While some mutations in EFL1 or SBDS result in insufficient proteins to meet the cell production of mature large ribosomal subunits, others do not affect the expression levels with unclear molecular defects. We studied the functional consequences of one such mutation using Saccharomyces cerevisiae Efl1 R1086Q, equivalent to human EFL1 R1095Q described in SDS patients. We characterised the enzyme kinetics and energetic basis outlining the recognition of this mutant to guanine nucleotides and Sdo1, and their interplay in solution. From our data, we propose a model where the conformational change in Efl1 depends on a long-distance network of interactions that are disrupted in mutant R1086Q, whereby Sdo1 and the guanine nucleotides no longer elicit the conformational changes previously described in the wild-type protein. These findings point to the molecular malfunction of an EFL1 mutant and its possible impact on SDS pathology.

Energetic and structural effects of the Tanford transition on ligand recognition of bovine ß-lactoglobulin.

Bovine ß-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of ß-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

Influence of physicochemical factors on environmental availability and distribution of semiochemicals that affect Varroa destructor and phylogenetically close organisms: classification by VHWOC PCA-clustering.

Varroa destructor parasites Apis mellifera larvae following the interception of the semiochemicals involved in bee communication; thus, the semiochemical availability and distribution pathways take place within different physicochemical environments. The structure of 172 molecules with semiochemical activity on Varroa destructor was used to compute the representative physicochemical descriptors of the thermodynamic partition among different physicochemical environments: vapor pressure (V), Henry's coefficient (H), water solubility constant (W), octanol-water partition coefficient (O) and organic carbon partition coefficient (C); VHWOC. The principal component analysis (PCA) and hierarchical clustering of VHWOC descriptors allowed us to establish the trend in availability and distribution of the semiochemicals resulting in a 4 classes model of physicochemical environments: Class 1, Soluble/Volatile; Class 2, Soluble; Class 3, Contact; Class 4, Adsorbed/Volatile. Our results suggest that semiochemicals can transit between different thermodynamic equilibrium phases depending on environment conditions. The classification prediction of the model was tested on 6 new molecules obtained from ketonic extracts of L5 Apis mellifera drone larvae; locating them in class 4, which was consistent with their molecular structure. This study can be the starting point for the design of synthetic semiochemicals or for the control of Varroa destructor. In addition, the method can be used in the analysis of other semiochemical groups.

Ligand binding and self-association cooperativity of ß-lactoglobulin.

Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self-association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine ß-lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled-equilibria revealed that dimerization exerts disparate effects on the ligand-binding capacity of ß-lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of ß-lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution.