Fine-tuning of the Hsc70-based Human Protein Disaggregase Machinery by the Distinctive C-terminal Extension of Apg2.

Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). Together with DnaJB1, Apg2 serves to nucleate Hsc70 molecules into sites where productive entropic pulling forces can be developed. During aggregate reactivation, Apg2 performs as a specialized nucleotide exchange factor, but the origin of its specialization is poorly defined. Here we report on the role of the distinctive C-terminal extension present in Apg2 and other metazoan homologs. We found that the first part of this Apg2 subdomain, with propensity to adopt α-helical structure, interacts with the nucleotide binding domain of Hsc70 in a nucleotide-dependent manner, contributing significantly to the stability of the Hsc70:Apg2 complex. Moreover, the second intrinsically disordered segment of Apg2 C-terminal extension plays an important role as a downregulator of nucleotide exchange. An NMR analysis showed that the interaction with Hsc70 nucleotide binding domain modifies the chemical environment of residues located in important functional sites such as the interface between lobe I and II and the nucleotide binding site. Our data indicate that Apg2 C-terminal extension is a fine-tuner of human Hsc70 activity that optimizes the substrate remodeling ability of the chaperone system.

The Isolation of New Pore-Forming Toxins from the Sea Anemone Actinia fragacea Provides Insights into the Mechanisms of Actinoporin Evolution.

Random mutations and selective pressure drive protein adaptation to the changing demands of the environment. As a consequence, nature favors the evolution of protein diversity. A group of proteins subject to exceptional environmental stress and known for their widespread diversity are the pore-forming hemolytic proteins from sea anemones, known as actinoporins. In this study, we identified and isolated new isoforms of actinoporins from the sea anemone Actinia fragacea (fragaceatoxins). We characterized their hemolytic activity, examined their stability and structure, and performed a comparative analysis of their primary sequence. Sequence alignment reveals that most of the variability among actinoporins is associated with non-functional residues. The differences in the thermal behavior among fragaceatoxins suggest that these variability sites contribute to changes in protein stability. In addition, the protein-protein interaction region showed a very high degree of identity (92%) within fragaceatoxins, but only 25% among all actinoporins examined, suggesting some degree of specificity at the species level. Our findings support the mechanism of evolutionary adaptation in actinoporins and reflect common pathways conducive to protein variability.

Regulation of Human Hsc70 ATPase and Chaperone Activities by Apg2: Role of the Acidic Subdomain.

Protein aggregate reactivation in metazoans is accomplished by the combined activity of Hsp70, Hsp40 and Hsp110 chaperones. Hsp110s support the refolding of aggregated polypeptides acting as specialized nucleotide exchange factors of Hsp70. We have studied how Apg2, one of the three human Hsp110s, regulates the activity of Hsc70 (HspA8), the constitutive Hsp70 in our cells. Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70, binding of the chaperone to protein aggregates and the refolding activity of the system, while it inhibits these three processes at high concentration. When the acidic subdomain of Apg2, a characteristic sequence present in the substrate binding domain of all Hsp110s, is deleted, the detrimental effects occur at lower concentration and are more pronounced, which concurs with an increase in the affinity of the Apg2 mutant for Hsc70. Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. In this line, the acidic subdomain might serve as a conformational switch to support dissociation of the Hsc70:Apg2 complex.

Functional characterization of Val60, a key residue involved in the membrane-oligomerization of fragaceatoxin C, an actinoporin from Actinia fragacea.

Actinoporins are pore-forming toxins produced by different sea anemones that self-assemble within the membranes of their target cells and compromise their function as a permeability barrier. The recently published three-dimensional structures of two oligomeric complexes formed by fragaceatoxin C point to Val60 as a key residue involved in the oligomerization of the functional pore. To gain insight into the mechanism of toxin oligomerization, different point mutations have been introduced at this position. Functional characterization of the muteins suggests that Val60 represents a hot-spot where the introduction of mutations hinders protein assembly and reduces the overall affinity for membranes.

Purification, cloning and characterization of fragaceatoxin C, a novel actinoporin from the sea anemone Actinia fragacea.

Actinia fragacea is commonly called the "strawberry" anemone because of the distinctive yellow or green spots displayed on its red column. Its venom contains several haemolytic proteins with a molecular mass of approximately 20 kDa that can be separated by ion-exchange column chromatography. One of them was purified to homogeneity and was named fragaceatoxin C (FraC). Its 15 N-terminal residues were identified by Edman degradation and served to obtain its complete DNA coding sequence by RT-PCR. The coding region of FraC was amplified and cloned in the expression vector pBAT-4. Purified recombinant FraC consists of 179 amino acids and multiple sequence alignment with other actinoporins clearly indicates that FraC belongs to this protein family. The secondary structure, thermal stability and lytic activity of native and recombinant FraC were practically identical and exhibit the same basic features already described for equinatoxin-II and sticholysin-II.

Par j 1 and Par j 2, the two major allergens in Parietaria judaica, bind preferentially to monoacylated negative lipids.

Par j 1 and Par j 2 proteins are the two major allergens in Parietaria judaica pollen, one of the main causes of allergic diseases in the Mediterranean area. Each of them contains eight cysteine residues organized in a pattern identical to that found in plant nonspecific lipid transfer proteins. The 139- and 102-residue recombinant allergens, corresponding respectively to Par j 1 and Par j 2, refold properly to fully functional forms, whose immunological properties resemble those of the molecules purified from the natural source. Molecular modeling shows that, despite the lack of extensive primary structure homology with nonspecific lipid transfer proteins, both allergens contain a hydrophobic cavity suited to accommodate a lipid ligand. In the present study, we present novel evidence for the formation of complexes of these natural and recombinant proteins from Parietaria pollen with lipidic molecules. The dissociation constant of oleyl-lyso-phosphatidylcholine is 9.1 +/- 1.2 microm for recombinant Par j 1, whereas pyrenedodecanoic acid shows a much higher affinity, with a dissociation constant of approximately 1 microm for both recombinant proteins, as well as for the natural mixture. Lipid binding does not alter the secondary structure content of the protein but is very efficient in protecting disulfide bonds from reduction by dithiothreitol. We show that Par j 1 and Par j 2 not only bind lipids from micellar dispersions, but also are able to extract and transfer negative phospholipids from bilayers.

Hydrogen-exchange stability analysis of Bergerac-Src homology 3 variants allows the characterization of a folding intermediate in equilibrium.

Amide hydrogendeuterium exchange rates have been determined for two mutants of alpha-spectrin Src homology 3 domain (WT), containing an elongated stable (SHH) and unstable (SHA) distal loop. SHA, similarly to WT, follows a two-state transition, whereas SHH apparently folds via a three-state mechanism. Native-state amide hydrogen exchange is effective in ascribing energetic readjustments observed in kinetic experiments to species stabilized within the denatured base and distinguishing those from high-energy barrier crossings. Comparison of DeltaG(ex) and m(ex) parameters for amide protons of these mutants demonstrates the existence of an intermediate and allows the identification of protons protected in this state. The consolidation of a form containing a prefolded long beta-hairpin induces the switch to a three-state mechanism in an otherwise two-state folder. It can be inferred that the unbalanced high stability of individual elements of secondary structure in a polypeptide could ultimately complicate its folding mechanism.

Unspecific hydrophobic stabilization of folding transition states.

Here we present a method for determining the inference of non-native conformations in the folding of a small domain, alpha-spectrin Src homology 3 domain. This method relies on the preservation of all native interactions after Tyr/Phe exchanges in solvent-exposed, contact-free positions. Minor changes in solvent exposure and free energy of the denatured ensemble are in agreement with the reverse hydrophobic effect, as the Tyr/Phe mutations slightly change the polypeptide hydrophilic/hydrophobic balance. Interestingly, more important Gibbs energy variations are observed in the transition state ensemble (TSE). Considering the small changes induced by the H/OH replacements, the observed energy variations in the TSE are rather notable, but of a magnitude that would remain undetected under regular mutations that alter the folded structure free energy. Hydrophobic residues outside of the folding nucleus contribute to the stability of the TSE in an unspecific nonlinear manner, producing a significant acceleration of both unfolding and refolding rates, with little effect on stability. These results suggest that sectors of the protein transiently reside in non-native areas of the landscape during folding, with implications in the reading of phi values from protein engineering experiments. Contrary to previous proposals, the principle that emerges is that non-native contacts, or conformations, could be beneficial in evolution and design of some fast folding proteins.