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Understanding the Frustration Arising from the Competition Between Function, Misfolding, and Aggregation in a Globular Protein

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Understanding the Frustration Arising from the Competition Between Function, Misfolding, and Aggregation in a Globular Protein Understanding the frustration arising from the competition between function, misfolding, and aggregation in a globular protein Stefano Giannia,b,1,2, Carlo Camillonib,1, Rajanish Giria, Angelo Totoa, Daniela Bonettia, Angela Morronea, Pietro Sormannib, Maurizio Brunoria, and Michele Vendruscolob,2 aDipartimento di Scienze Biochimiche A. Rossi Fanelli, Istituto Pasteur–Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del Consiglio Nazionale delle Ricerche, Università di Roma La Sapienza, 00185 Rome, Italy; and bDepartment of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom Edited* by Peter G. Wolynes, Rice University, Houston, TX, and approved August 20, 2014 (received for review March 20, 2014) Folding and function may impose different requirements on the complex with the two main components of the iron–sulfur cluster amino acid sequences of proteins, thus potentially giving rise to biogenesis machinery (8–11). This protein offers good opportuni- conflict. Such a conflict, or frustration, can result in the formation of ties for investigating the relationships between folding, misfolding, partially misfolded intermediates that can compromise folding and and disease. Indeed, its dysfunction is related to a neurodegener- promote aggregation. We investigate this phenomenon by studying ative disease called Friedreich’s ataxia (12). Frataxin is also capa- frataxin, a protein whose normal function is to facilitate the for- ble of binding different divalent and trivalent cations, whose mation of iron–sulfur clusters but whose mutations are associated recognition sites have been mapped (13). Furthermore, frataxin with Friedreich’s ataxia. To characterize the folding pathway of this is involved in donating iron to ferrochelatease via direct inter- protein we carry out a Φ-value analysis and use the resulting struc- action through an extended binding site involving some of the tural information to determine the structure of the folding transi- residues implicated in metal binding (14). tion state, which we then validate by a second round of rationally We have previously shown that frataxin folds via a complex designed mutagenesis. The analysis of the transition-state structure mechanism, which we described through a broad free energy reveals that the regions involved in the folding process are highly barrier (15). This feature, which has been associated with frus- aggregation-prone. By contrast, the regions that are functionally tration (16), allows the experimental characterization of both the important are partially misfolded in the transition state but highly early and late events of folding (16–19). In this work we explored resistant to aggregation. Taken together, these results indicate that in the mechanistic details of the folding reaction of frataxin at frataxin the competition between folding and function creates the residue-level resolution. This result was achieved by character- possibility of misfolding, and that to prevent aggregation the amino izing the structures of both the early and late events of folding acid sequence of this protein is optimized to be highly resistant to using Φ-value analysis (20) and restrained molecular dynamics aggregation in the regions involved in misfolding. simulations (21). By analyzing the structures of the different states along the folding process we found an unexpected number rustration is a general condition that arises in the presence of of nonnative interactions that slow down folding and superpose Fconflicting requirements. A system is frustrated when it is with the highly frustrated regions, as detected by the frustratometer impossible to fully minimize its energy by optimizing simulta- server (22). The nonnative regions, which display peculiar Φ values, neously all of the possible interactions among its components either negative or greater than unity, were predicted on the basis of (1). Although complex systems tend in general to exhibit frus- the transition state structures determined from the Φ values, tration because of the large number and heterogeneity of their and subsequently confirmed by a second round of amino acid components, protein molecules are remarkable in that their folding process involves interactions that express a minimal Significance level of frustration. According to the so-called principle of mini- mal frustration, the energy of proteins decreases as they explore conformations increasingly similar in structure to the native state The amino acid sequence of a protein encodes a wide range of (2). Consequently, the free energy landscape of proteins is char- different properties, including function, folding, and avoidance acterized by the presence of a well-defined global minimum and of aggregation. The resulting requirements on the sequence may be in conflict with each other, thus creating the possibility very few other local minima, which are typically intermediate of misfolding and dysfunction. To investigate possible mecha- states along the folding pathway. This organization of confor- nisms whereby such unwanted outcomes can be prevented, mational space normally ensures rapid and reliable folding (2–6). we study the folding process of frataxin, a protein whose ab- Proteins, however, have evolved not only to fold, but also to errant folding is associated with Friedreich’s ataxia. Our results function. Because the evolutionary constraints that select for a indicate that the regions prone to misfolding are highly given function may be in conflict with the folding process, it is protected against aggregation along the folding pathway of possible that local frustration patterns may localize in specific this protein. regions of proteins, in particular in their active sites. Indeed, a statistical survey of different proteins has shown that frustrated Author contributions: S.G., C.C., M.B., and M.V. designed research; C.C., R.G., A.T., D.B., A.M., BIOPHYSICS AND interactions tend to cluster at binding sites and that such frus- and P.S. performed research; S.G., C.C., R.G., A.T., D.B., A.M., P.S., M.B., and M.V. analyzed COMPUTATIONAL BIOLOGY tration decreases upon complex formation (7). Because frustra- data; and S.G., M.B., and M.V. wrote the paper. tion is associated with the presence of local minima in the free The authors declare no conflict of interest. energy landscape, it is important to understand how proteins *This Direct Submission article had a prearranged editor. have evolved to minimize the possible effects associated with these 1S.G. and C.C. contributed equally to this work. local minima, which are likely to contain misfolded elements and 2To whom correspondence may be addressed. Email: [email protected] or thus to potentially give rise to aggregation. [email protected]. To address this question we studied frataxin, a mitochondrial This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 2+ 3+ protein that binds both Fe and Fe ions and forms a ternary 1073/pnas.1405233111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1405233111 PNAS | September 30, 2014 | vol. 111 | no. 39 | 14141–14146 Downloaded by guest on September 28, 2021 substitutions rationally designed to probe misfolded regions analysis of the observed folding and unfolding amplitudes for along the folding pathway. frataxin (Fig. S2) confirms the absence of burst-phase intermediates The characterization of the folding pathway of frataxin and of lost in the dead time of the stopped flow, which is consistent with its misfolded elements enables us to discuss the competition earlier suggestions of a broad-barrier model (15). between folding and function and its consequences for misfold- In agreement with previous observations on wild-type frataxin ing and aggregation. (15), all mutants displayed a pronounced curvature in both the folding and unfolding arms of the chevron plot. Different muta- Results tions, however, had distinct effects on the folding and unfolding Φ-Value Analysis. We performed Φ-value analysis (20) and restrained rate constants and on the curvature of the chevron plots. For molecular dynamics simulations (21) to characterize the folding example, the A26G, L33A, L81A, and Y68A mutants display pathway of yeast frataxin at residue-level resolution. Taking ad- nearly V-shaped chevron plots, with Y68A showing the onset of vantage of the characteristic folding of frataxin, which involves a an upward curvature. By contrast, the L37A and V115A mutants broad free energy barrier, Φ values were experimentally mea- display a degree of curvature more pronounced than that of wild- sured for early, intermediate, and late events along the folding type frataxin. These positions are in direct contact in the native pathway, by following a methodology previously described (17) state, forming a major portion of its hydrophobic nucleus. Con- and briefly summarized below. sequently, we analyzed the data by following a model involving Twenty-one site-directed mutants of frataxin were designed, a broad free energy barrier (17). According to this model, a com- expressed, and purified (Fig. 1 and Table S1). One of these mutants plexity in the chevron plot arises from progressive changes in expressed poorly and could not be analyzed; the remaining mutants structure of the transition state as the native state is destabilized were subjected to kinetic folding studies and 18 Φ values could (23–25). If the transition state can be associated with a broad be calculated. Kinetic folding experiments were performed
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