Generic structures of cytotoxic liprotides: nano-sized complexes with oleic acid cores and shells of disordered proteins.

The cytotoxic complex formed between α-lactalbumin and oleic acid (OA) has inspired many studies on protein-fatty acid complexes, but structural insight remains sparse. After having used small-angle X-ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein-oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core-shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off-loading of the OA into cell membranes.

Low-resolution structures of OmpA⋠DDM protein-detergent complexes.

We have used SAXS to determine the low-resolution structure of the outer-membrane protein OmpA from E. coli solubilized by the surfactant dodecyl maltoside (DDM). We have studied three variants of the transmembrane domain of OmpA-namely monomers, self-associated dimers, and covalently linked dimers-as well as the monomeric species of the full-length protein with the periplasmic domain. We can successfully model the structures of the monomeric and covalently linked dimer as one and two natively folded proteins in a DDM micelle, respectively, whereas the noncovalently linked dimer presents a more complicated structure, possibly due to higher-order species. We have determined the structure of the full-length protein to be that of a globular periplasmic domain attached through a flexible linker to the transmembrane domain. This approach provides valuable information about how membrane proteins are embedded in amphiphilic environments.

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein.

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.

Structure of the Mycobacterium tuberculosis cPknF and conformational changes induced in forkhead-associated regulatory domains.

Mycobacterium tuberculosis (Mtb) has 11 Serine-Threonine Protein Kinases (STPK) that control numerous physiological processes, including cell growth, cell division, metabolic flow, and transcription. PknF is one of the 11 Mtb STPKs that has, among other substrates, two FHA domains (FHA-1 and FHA-2) of the ATP-Binding Cassette (ABC) transporter Rv1747. Phosphorylation in T152 and T210 located in a non-structured linker that connects Rv1747 FHA domains is considerate to be the regulatory mechanism of the transporter. In this work, we resolved the three-dimensional structure of the PknF catalytic domain (cPknF) in complex with the human kinase inhibitor IKK16. cPknF is conserved when compared to other STPKs but shows specific residues in the binding site where the inhibitor is positioned. In addition, using Small Angle X-Ray Scattering analysis we monitored the behavior of the wild type and three FHA-phosphomimetic mutants in solution, and measured the cPknF affinity for these domains. The kinase showed higher affinity for the non-phosphorylated wild type domain and preference for phosphorylation of T152 inducing the rapprochement of the domains and significant structural changes. The results shed some light on the process of regulating the transporter's activity by phosphorylation and arises important questions about evolution and importance of this mechanism for the bacillus.