Understanding a Core Pilin of the Type IVa Pili of Acidithiobacillus thiooxidans, PilV.

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Improved Stability of Human CGI-58 Induced by Phosphomimetic S237E Mutation.

In lipolysis, the activating function of CGI-58 is regulated by its interaction with perilipin 1 (PLIN1) localized on the lipid droplet (LD), and its release is controlled by phosphorylation. Once lipolysis is stimulated by catecholamines, protein kinase A (PKA)-mediated phosphorylation enables the dissociation of the CGI-58/PLIN1 complex, thereby recruiting adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) to initiate fatty acid release. It has been shown that mouse CGI-58 mutant S239E, which mimics the phosphorylation of this residue, is able to dissociate from the CGI-58/PLIN1 complex and activate ATGL. Here, we analyze the stabilizing effect on human CGI-58 of a triple tryptophan to alanine mutant (3WA) on the LD-binding motif, as well as a quadruple mutant in which the phosphomimetic S237E substitution was introduced to the 3WA construct (3WA/S237E). We found that tryptophan residues promote wild-type (WT) protein aggregation in solution since their substitution for alanine residues favors the presence of the monomer. Our experimental data showed increased thermal stability and solubility of 3WA/S237E protein compared to the 3WA mutant. Moreover, the 3WA/S237E protein showed proper folding and a functional binding site for oleoyl-CoA. The analysis of a bioinformatic three-dimensional (3D) model suggests an intramolecular interaction between the phosphomimetic glutamic acid and a residue of the α/ß hydrolase core. This could explain the increased solubility and stability observed in the 3WA/S237E mutant and evidences the possible role of serine 237 phosphorylation.

Molecular ruler mechanism and interfacial catalysis of the integral membrane acyltransferase PatA.

Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity "ruler." We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acyl­coenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors.

Bioinformatic Analysis and Biophysical Characterization Reveal Structural Disorder in G0S2 Protein.

G0S2 is a small protein of 103 residues in length that is involved in multiple cellular processes. To date, several reports have shown that G0S2 functions by making direct protein-protein interactions with key proteins. In lipolysis, G0S2 specifically interacts with adipose triglyceride lipase, inhibiting its activity and resulting in lipolysis being downregulated. In a similar way, G0S2 also participates in the regulation of apoptosis, cell proliferation, and oxidative phosphorylation; however, information regarding G0S2 structural and biophysical properties is limited. In this work, we conducted a comparative structural analysis of human and mouse G0S2 proteins. Bioinformatics suggests the presence of a disordered C-terminal region in human G0S2. Experimental characterization by size-exclusion chromatography and dynamic light scattering showed that human and mouse G0S2 have different hydrodynamic properties. In comparison to the mouse G0S2, which behaves similar to a globular protein, the human G0S2 shows an elongated conformation, most likely by displaying a disordered C-terminal region. Further analysis of hydrodynamic properties under denaturing conditions suggests the presence of a structural element in the mouse protein that undergoes an order to disorder transition at low urea concentration. Structural analysis by circular dichroism revealed that in native conditions, both proteins are mainly unstructured, showing the presence of beta sheet structures. Further analysis of CD data suggests that both proteins belong to the premolten globule family of intrinsically disordered proteins. We suggest that the intrinsic disorder observed in the G0S2 protein may facilitate its interaction with multiple partners in the regulation of cellular metabolism.

Nucleotide Binding in an Engineered Recombinant Ca2+-ATPase N-Domain.

A recombinant Ca2+-ATPase nucleotide binding domain (N-domain) harboring the mutations Trp552Leu and Tyr587Trp was expressed and purified. Chemical modification by N-bromosuccinimide and fluorescence quenching by acrylamide showed that the displaced Trp residue was located at the N-domain surface and slightly exposed to solvent. Guanidine hydrochloride-mediated N-domain unfolding showed the low structural stability of the α6-loop-α7 motif (the new Trp location) located near the nucleotide binding site. The binding of nucleotides (free and in complex with Mg2+) to the engineered N-domain led to significant intrinsic fluorescence quenching (ΔFmax ∼ 30%) displaying a saturable hyperbolic pattern; the calculated affinities decreased in the following order: ATP > ADP = ADP-Mg2+ > ATP-Mg2+. Interestingly, it was found that Ca2+ binds to the N-domain as monitored by intrinsic fluorescence quenching (ΔFmax ∼ 12%) with a dissociation constant (Kd) of 50 µM. Notably, the presence of Ca2+ (200 µM) increased the ATP and ADP affinity but favored the binding of ATP over that of ADP. In addition, binding of ATP to the N-domain generated slight changes in secondary structure as evidenced by circular dichroism spectral changes. Molecular docking of ATP to the N-domain provided different binding modes that potentially might be the binding stages prior to γ-phosphate transfer. Finally, the nucleotide binding site was studied by fluorescein isothiocyanate labeling and molecular docking. The N-domain of Ca2+-ATPase performs structural dynamics upon Ca2+ and nucleotide binding. It is proposed that the increased affinity of the N-domain for ATP mediated by Ca2+ binding may be involved in Ca2+-ATPase activation under normal physiological conditions.