WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.
The regulation and localization of signaling enzymes is often mediated by accessory modular domains, which frequently function in tandems. The ability of these tandems to adopt multiple conformations is as important for proper regulation as the individual domain specificity. A paradigmatic example is Abl, a ubiquitous tyrosine kinase of significant pharmacological interest. SH3 and SH2 domains inhibit Abl by assembling onto the catalytic domain, allosterically clamping it in an inactive state. We investigate the dynamics of this SH3-SH2 tandem, using microsecond all-atom simulations and differential scanning calorimetry. Our results indicate that the Abl tandem is a two-state switch, alternating between the conformation observed in the structure of the autoinhibited enzyme and another configuration that is consistent with existing scattering data for an activated form. Intriguingly, we find that the latter is the most probable when the tandem is disengaged from the catalytic domain. Nevertheless, an amino acid stretch preceding the SH3 domain, the so-called N-cap, reshapes the free-energy landscape of the tandem and favors the interaction of this domain with the SH2-kinase linker, an intermediate step necessary for assembly of the autoinhibited complex. This allosteric effect arises from interactions between N-cap and the SH2 domain and SH3-SH2 connector, which involve a phosphorylation site. We also show that the SH3-SH2 connector plays a determinant role in the assembly equilibrium of Abl, because mutations thereof hinder the engagement of the SH2-kinase linker. These results provide a thermodynamic rationale for the involvement of N-cap and SH3-SH2 connector in Abl regulation and expand our understanding of the principles of modular domain organization.allosteric inhibitors | protein-protein interactions | macromolecular assembly | population shift | leukemia
The amino acid sequence of Leishmania mexicana triose phosphate isomerase is unique in having at position 65 a glutamic acid instead of a glutamine. The stability properties of LmTIM and the E65Q mutant were investigated by pH and guanidinium chloride-induced unfolding. The crystal structure of E65Q was determined. Three important observations were made: (a) there are no structural rearrangements as the result of the substitution; (b) the mutant is more stable than the wild-type; and (c) the stability of the wild-type enzyme shows strong pH dependence, which can be attributed to the ionization of Glu65. Burying of the Glu65 side chain in the uncharged environment of the dimer interface results in a shift in pK a of more than 3 units. The pH-dependent decrease in overall stability is due to weakening of the monomer±monomer interactions (in the dimer). The E65Q substitution causes an increase in stability as the result of the formation of an additional hydrogen bond in each subunit (DDG 8 of 2 kcal´mol 21 per monomer) and the elimination of a charged group in the dimer interface (DDG 8 of at least 9 kcal´mol 21 per dimer). The computated shift in pK a and the stability of the dimer calculated from the charge distribution in the protein structure agree closely with the experimental results.The guanidinium chloride dependence of the unfolding constant was smaller than expected from studies involving monomeric model proteins. No intermediates could be identified in the unfolding equilibrium by combining fluorescence and CD measurements. Study of a stable monomeric triose phosphate isomerase variant confirmed that the phenomenon persists in the monomer.Keywords: Leishmania mexicana; pK a calculations; stability; triose phosphate isomerase.Burying a titratable group in a protein without an oppositely charged group nearby has been shown to change the pK a value of the charged group substantially [1]. Several examples of pK a shifts being related to the overall stability of proteins have been reported [2±7].Leishmania mexicana triose phosphate isomerase (LmTIM) is unique because it contains a substitution in an otherwise conserved sequence (64±66) located at the beginning of loop 3, namely Q65E [8]. In the related trypanosomal TIM (TbTIM) Gln65 is completely buried in the dimer interface and participates in an intersubunit hydrogen-bond network [9±10]. From the crystal structure of LmTIM, it was evident that no major structural rearrangements had taken place in the vicinity of the Glu65 side chain [11] and that Glu65 was protonated. Substituting Glu65 in LmTIM for a Gln yielded a protein with considerably greater thermostability than the wild-type. Moreover, the mutation abolished the pH dependence (pH 5±9) of the thermostability of wild-type LmTIM, indicating that the ionization of Glu65 has a significant impact on the overall stability of the dimer [11].The availability of the E65Q mutant and the high-resolution crystal structure of LmTIM provided us with a system that is eminently suited to the study of the effects of...
We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of ca-helix and 8-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain. The growth of structure in the small module is highly cooperative and does not result from the hierarchical accretion of substructures.Proteins are synthesized from the N terminus in vivo. The information contained in the amino acid sequence encodes the three-dimensional structure of the biologically active protein (1) so that many proteins spontaneously fold to their correct conformations. Accessory proteins are found in vivo, however, which appear to prevent off-pathway reactions, such as aggregation (2-4), but the final folded structure of the protein is still dictated by the amino acid sequence. Certain accessory proteins interact with nascent incompletely folded polypeptides. The interactions may possibly be dictated by the structural characteristics of the growing chain as it emerges from the ribosome (5-7).Most of our current detailed knowledge about protein folding is derived from studies in vitro, usually from the refolding of denatured mature polypeptides (8,9). Studies in vivo on the development of structure in a growing polypeptide chain during biosynthesis are too difficult at the molecular level, although pioneering characterization has been attempted by using the binding of antibodies raised against the native structure (10). A parallel approach to the problem is to study the polypeptide chain as it is synthesized in vitro under controlled conditions in the absence of complicating factors. Such a study could answer the following questions. What happens to a polypeptide chain as it is synthesized from the N terminus in vitro? Do stable structures form early in synthesis? When does recognizable native secondary structure form? Do subsets of structures form and progressively assemble in a hierarchical manner?Here we examine the development of structure in a small protein, whose refolding pathway in vitro has been described in great detail (11)(12)(13), by physically dissecting it into N-terminal fragments of increasing length. The protein chosen is the 64-residue chymotrypsin inhibitor 2 (CI-2) from barley seeds because it approaches the simplest system possible and may represent a minimal folding unit comparable to a single domain (module) in a larger protein: it is a very small monomer that consists of a single domain (module) without disulfide crosslinks that constrain the unfolded state, and it has no complications from the presence of cis peptidyl-prolylThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisem...
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