Surfactant protein B (SP-B) is essential in transferring surface-active phospholipids from membrane-based surfactant complexes into the alveolar air-liquid interface. This allows maintaining the mechanical stability of the surfactant film under high pressure at the end of expiration; therefore, SP-B is crucial in lung function. Despite its necessity, the structure and the mechanism of lipid transfer by SP-B have remained poorly characterized. Earlier, we proposed higher-order oligomerization of SP-B into ring-like supramolecular assemblies. In the present work, we used coarse-grained molecular dynamics simulations to elucidate how the ring-like oligomeric structure of SP-B determines its membrane binding and lipid transfer. In particular, we explored how SP-B interacts with specific surfactant lipids, and how consequently SP-B reorganizes its lipid environment to modulate the pulmonary surfactant structure and function. Based on these studies, there are specific lipid-protein interactions leading to perturbation and reorganization of pulmonary surfactant layers. Especially, we found compelling evidence that anionic phospholipids and cholesterol are needed or even crucial in the membrane binding and lipid transfer function of SP-B. Also, on the basis of the simulations, larger oligomers of SP-B catalyze lipid transfer between adjacent surfactant layers. Better understanding of the molecular mechanism of SP-B will help in the design of therapeutic SP-B-based preparations and novel treatments for fatal respiratory complications, such as the acute respiratory distress syndrome.
- MeSH
- Phospholipids chemistry MeSH
- Protein Conformation MeSH
- Lipid Bilayers chemistry metabolism MeSH
- Models, Molecular MeSH
- Protein Multimerization MeSH
- Pulmonary Surfactants chemistry MeSH
- Pulmonary Surfactant-Associated Protein B chemistry metabolism MeSH
- Molecular Dynamics Simulation MeSH
- Binding Sites MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.
Mason-Pfizer monkey virus (M-PMV) Gag protein contains a domain p12 that is unique to this virus (simian retrovirus-3) and its close relatives. The alpha-helical N-terminal half of p12, which contains a leucine zipper-like region, forms ordered structures in E. coli and the C-terminal half can form SDS-resistant oligomers in vitro. Together these properties suggest that p12 is a strong protein-protein interaction domain that facilitates Gag-Gag oligomerization. We have analyzed the oligomerization potential of a panel of p12 mutants, including versions containing substituted dimer, trimer, and tetramer leucine zippers, expressed in bacteria and in the context of the Gag precursor expressed in vitro and in cells. Purified recombinant p12 and its mutants could form various oligomers as shown by chemical cross-linking experiments. Within Gag these same mutants could assemble when overexpressed in cells. In contrast, all the mutants, including the leucine zipper mutants, were assembly defective in a cell-free system. These data highlight the importance of a region containing alternating leucines and isoleucines within p12, but also indicate that this domain's scaffold-like function is more complex than small number oligomerization.
- MeSH
- Chlorocebus aethiops MeSH
- COS Cells MeSH
- Financing, Organized MeSH
- Gene Products, gag genetics metabolism MeSH
- Leucine Zippers MeSH
- Mason-Pfizer monkey virus physiology genetics ultrastructure MeSH
- Molecular Sequence Data MeSH
- Mutation MeSH
- Recombinant Proteins genetics isolation & purification metabolism MeSH
- Protein Structure, Tertiary MeSH
- Microscopy, Electron, Transmission MeSH
- Protein Binding MeSH
- Virion ultrastructure MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used to study the oligomeric states of soluble proteins. A mild binding of the used protein stains to nondissociated protein oligomers imposed a charge shift on the proteins resulting into separation of protein species according to their size under physiological conditions. Adsorbed stains could be easily removed after electrophoresis by washing of polyacrylamide gel with buffer and protein complexes could be visualized either by the detection of their enzyme activity or by using a nonspecific protein stain. The specific detection of enzyme activity of glycosidases, lactate dehydrogenase, or phosphatases was shown as an example.
Protein-protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8-Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.
- MeSH
- Photochemical Processes MeSH
- Humans MeSH
- Protein Interaction Mapping methods MeSH
- Models, Molecular MeSH
- Protein Multimerization MeSH
- 14-3-3 Proteins chemistry metabolism MeSH
- Amino Acid Sequence MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Degradation of the plant hormone cytokinin is controlled by cytokinin oxidase/dehydrogenase (CKX) enzymes. The molecular and cellular behavior of these proteins is still largely unknown. In this study, we show that CKX1 is a type II single-pass membrane protein that localizes predominantly to the endoplasmic reticulum (ER) in Arabidopsis (Arabidopsis thaliana). This indicates that this CKX isoform is a bona fide ER protein directly controlling the cytokinin, which triggers the signaling from the ER. By using various approaches, we demonstrate that CKX1 forms homodimers and homooligomers in vivo. The amino-terminal part of CKX1 was necessary and sufficient for the protein oligomerization as well as for targeting and retention in the ER. Moreover, we show that protein-protein interaction is largely facilitated by transmembrane helices and depends on a functional GxxxG-like interaction motif. Importantly, mutations rendering CKX1 monomeric interfere with its steady-state localization in the ER and cause a loss of the CKX1 biological activity by increasing its ER-associated degradation. Therefore, our study provides evidence that oligomerization is a crucial parameter regulating CKX1 biological activity and the cytokinin concentration in the ER. The work also lends strong support for the cytokinin signaling from the ER and for the functional relevance of the cytokinin pool in this compartment.
- MeSH
- Arabidopsis metabolism MeSH
- Endoplasmic Reticulum metabolism MeSH
- Membrane Proteins chemistry metabolism MeSH
- Protein Multimerization * MeSH
- Oxidoreductases chemistry metabolism MeSH
- Protein Domains MeSH
- Protein Sorting Signals MeSH
- Arabidopsis Proteins chemistry metabolism MeSH
- Recombinant Fusion Proteins metabolism MeSH
- Amino Acid Sequence MeSH
- Protein Stability MeSH
- Green Fluorescent Proteins metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.
- MeSH
- Phosphorylation MeSH
- Caspase 2 chemistry genetics metabolism MeSH
- Protein Conformation * MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Models, Molecular * MeSH
- Protein Multimerization * MeSH
- Mutation MeSH
- Protein Isoforms genetics metabolism MeSH
- Protein Precursors chemistry genetics metabolism MeSH
- 14-3-3 Proteins chemistry genetics metabolism MeSH
- Recombinant Proteins chemistry metabolism MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.
- MeSH
- Amino Acid Motifs MeSH
- Aspartic Acid Proteases chemistry metabolism MeSH
- HEK293 Cells MeSH
- Conserved Sequence MeSH
- Crystallography, X-Ray MeSH
- Humans MeSH
- Scattering, Small Angle MeSH
- Protein Interaction Mapping MeSH
- Evolution, Molecular MeSH
- Models, Molecular MeSH
- Protein Multimerization MeSH
- Polyubiquitin metabolism MeSH
- Protein Domains MeSH
- Proteolysis MeSH
- Solutions MeSH
- Saccharomyces cerevisiae Proteins chemistry metabolism MeSH
- Saccharomyces cerevisiae metabolism MeSH
- Amino Acid Sequence MeSH
- Sequence Analysis, Protein MeSH
- Structural Homology, Protein * MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.
- MeSH
- Cysteine chemistry genetics metabolism MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Kinetics MeSH
- Protein Conformation MeSH
- Humans MeSH
- Models, Molecular MeSH
- Protein Multimerization * MeSH
- Mutation MeSH
- Computer Simulation MeSH
- 14-3-3 Proteins chemistry genetics metabolism MeSH
- Protein Stability MeSH
- Thermodynamics * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In current work, we used recombinant OspC protein derived from B. afzelii strain BRZ31 in the native homodimeric fold for mice immunization and following selection process to produce three mouse monoclonal antibodies able to bind to variable parts of up to five different OspC proteins. Applying the combination of mass spectrometry assisted epitope mapping and affinity based theoretical prediction we have localized regions responsible for antigen-antibody interactions and approximate epitopes' amino acid composition. Two mAbs (3F4 and 2A9) binds to linear epitopes located in previously described immunogenic regions in the exposed part of OspC protein. The third mAb (2D1) recognises highly conserved discontinuous epitope close to the ligand binding domain 1.
- MeSH
- Antigens, Bacterial chemistry genetics immunology MeSH
- Borrelia burgdorferi chemistry genetics immunology MeSH
- Epitope Mapping * MeSH
- Protein Multimerization * MeSH
- Mice, Inbred BALB C MeSH
- Antibodies, Monoclonal, Murine-Derived chemistry immunology MeSH
- Mice MeSH
- Bacterial Outer Membrane Proteins chemistry genetics immunology MeSH
- Protein Folding MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
