The armadillo-repeat domain of plakophilin 1 binds the C-terminal sterile alpha motif (SAM) of p73.

BACKGROUND: Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules. METHODS: We studied the binding of SAMp73 with the armadillo domain of PKP1 (ARM-PKP1) in the absence and the presence of 100 mM NaCl, by using several biophysical techniques, namely fluorescence, far-ultraviolet circular dichroism (CD), nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), and molecular docking and simulations. RESULTS: Association was observed between the two proteins, with a dissociation constant of ~5 µM measured by ITC and fluorescence in the absence of NaCl. The binding region of SAMp73 involved residues of the so-called "middle-loop-end-helix" binding region (i.e., comprising the third helix, together with the C terminus of the second one, and the N-cap of the fourth), as shown by 15N, 1H- HSQC-NMR spectra. Molecular modelling provided additional information on the possible structure of the binding complex. CONCLUSIONS: This newly-observed interaction could have potential therapeutic relevance in the tumour pathways where PKP1 is involved, and under conditions when there is a possible inactivation of p53. GENERAL SIGNIFICANCE: The discovery of the binding between SAMp73 and ARM-PKP1 suggests a functional role for their interaction, including the possibility that SAMp73 could assist PKP1 in signalling pathways.

The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability.

Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range.

Human importin α3 and its N-terminal truncated form, without the importin-ß-binding domain, are oligomeric species with a low conformational stability in solution.

BACKGROUND: Eukaryotic cells have a continuous transit of macromolecules between the cytoplasm and the nucleus. Several carrier proteins are involved in this transport. One of them is importin α, which must form a complex with importin ß to accomplish its function, by domain-swapping its 60-residue-long N terminus. There are several human isoforms of importin α; among them, importin α3 has a particularly high flexibility. METHODS: We studied the conformational stability of intact importin α3 (Impα3) and its truncated form, where the 64-residue-long, N-terminal importin-ß-binding domain (IBB) has been removed (ΔImpα3), in a wide pH range, with several spectroscopic, biophysical, biochemical methods and with molecular dynamics (MD). RESULTS: Both species acquired native-like structure between pH 7 and 10.0, where Impα3 was a dimer (with an apparent self-association constant of ~10 µM) and ΔImpα3 had a higher tendency to self-associate than the intact species. The acquisition of secondary, tertiary and quaternary structure, and the burial of hydrophobic patches, occurred concomitantly. Both proteins unfolded irreversibly at physiological pH, by using either temperature or chemical denaturants, through several partially folded intermediates. The MD simulations support the presence of these intermediates. CONCLUSIONS: The thermal stability of Impα3 at physiological pH was very low, but was higher than that of ΔImpα3. Both proteins were stable in a narrow pH range, and they unfolded at physiological pH populating several intermediate species. GENERAL SIGNIFICANCE: The low conformational stability explains the flexibility of Impα3, which is needed to carry out its recognition of complex cargo sequences.

Modulation of the potassium channel KcsA by anionic phospholipids: Role of arginines at the non-annular lipid binding sites.

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines.

Conformational plasticity in the KcsA potassium channel pore helix revealed by homo-FRET studies.

Potassium channels selectivity filter (SF) conformation is modulated by several factors, including ion-protein and protein-protein interactions. Here, we investigate the SF dynamics of a single Trp mutant of the potassium channel KcsA (W67) using polarized time-resolved fluorescence measurements. For the first time, an analytical framework is reported to analyze the homo-Förster resonance energy transfer (homo-FRET) within a symmetric tetrameric protein with a square geometry. We found that in the closed state (pH 7), the W67-W67 intersubunit distances become shorter as the average ion occupancy of the SF increases according to cation type and concentration. The hypothesis that the inactivated SF at pH 4 is structurally similar to its collapsed state, detected at low K+, pH 7, was ruled out, emphasizing the critical role played by the S2 binding site in the inactivation process of KcsA. This homo-FRET approach provides complementary information to X-ray crystallography in which the protein conformational dynamics is usually compromised.

The C Terminus of the Ribosomal-Associated Protein LrtA Is an Intrinsically Disordered Oligomer.

The 191-residue-long LrtA protein of Synechocystis sp. PCC 6803 is involved in post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family, intervening in protein synthesis. The protein consists of two domains: The N-terminal region (N-LrtA, residues 1⁻101), which is common to all the members of the HPF, and seems to be well-folded; and the C-terminal region (C-LrtA, residues 102⁻191), which is hypothesized to be disordered. In this work, we studied the conformational preferences of isolated C-LrtA in solution. The protein was disordered, as shown by computational modelling, 1D-¹H NMR, steady-state far-UV circular dichroism (CD) and chemical and thermal denaturations followed by fluorescence and far-UV CD. Moreover, at physiological conditions, as indicated by several biochemical and hydrodynamic techniques, isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. Thus, C-LrtA was an oligomeric disordered protein.

Selective exclusion and selective binding both contribute to ion selectivity in KcsA, a model potassium channel.

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1-S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+ These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity.

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA).

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp(67)-Glu(71)-Asp(80) inactivation triad within the channel structure and its bearing on the selectivity filter.

Detergent-labile, supramolecular assemblies of KcsA: relative abundance and interactions involved.

In this work, we illustrate the ability of the prokaryotic potassium channel KcsA to assemble into a variety of supramolecular clusters of defined sizes containing the tetrameric KcsA as the repeating unit. Such clusters, particularly the larger ones, are markedly detergent-labile and thus, disassemble readily upon exposure to the detergents commonly used in protein purification or conventional electrophoresis analysis. This is a reversible process, as cluster re-assembly occurs upon detergent removal and without the need of added membrane lipids. Interestingly, the dimeric ensemble between two tetrameric KcsA molecules are quite resistant to detergent disassembly to individual KcsA tetramers and along with the latter, are likely the basic building blocks through which the larger clusters are organized. As to the proteins domains involved in clustering, we have observed disassembly of KcsA clusters by SDS-like alkyl sulfates. As these amphiphiles bind to inter-subunit, "non-annular" sites on the protein, these observations suggest that such sites also mediate channel-channel interactions leading to cluster assembly.