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Structural Characterization of a Misfolded Intermediate Populated During the Folding Process of a PDZ Domain

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Structural Characterization of a Misfolded Intermediate Populated During the Folding Process of a PDZ Domain ARTICLES Structural characterization of a misfolded intermediate populated during the folding process of a PDZ domain Stefano Gianni1,4, Ylva Ivarsson1,3,4, Alfonso De Simone2, Carlo Travaglini-Allocatelli1, Maurizio Brunori1 & Michele Vendruscolo2 Incorrectly folded states transiently populated during the protein folding process are potentially prone to aggregation and have been implicated in a range of misfolding disorders that include Alzheimer’s and Parkinson’s diseases. Despite their importance, however, the structures of these states and the mechanism of their formation have largely escaped detailed characterization because of their short-lived nature. Here we present the structures of all the major states involved in the folding process of a PDZ domain, which include an off-pathway misfolded intermediate. By using a combination of kinetic, protein engineering, biophysical and computational techniques, we show that the misfolded intermediate is characterized by an alternative packing of the N-terminal b-hairpin onto an otherwise native-like scaffold. Our results suggest a mechanism of formation of incorrectly folded transient compact states by which misfolded structural elements are assembled together with more extended native-like regions. The large majority of biochemical processes that take place in living are small (90–100 amino acid) domains that adopt a six-stranded cells rely on the ability of proteins to fold into their functional native β-sandwich fold flanked by two α-helices22, with the typical linear states1. When folding fails, the identification and elimination of the arrangement of the secondary structure elements being βββαββαβ. misfolded species becomes a critical task, as the accumulation of mis- However, the PDZ domain family also comprises circularly permuted folded proteins is associated with a wide range of human disorders variants, whereby the canonical β1 strand is shifted to the C terminus such as Alzheimer’s and Parkinson’s diseases and type II diabetes2,3. of the sequence23,24. A number of canonical PDZ domains fold via a To distinguish between native and non-native conformations of pro- conserved three-state mechanism, proceeding through two sequential teins, the cell uses various sensor molecules, including molecular transition states and one unstable on-pathway intermediate25–30. By chaperones, that selectively recognize misfolded species4–6. Thus, contrast, the folding process of a naturally occurring, circularly per- the quality control system does not degrade functional species, but it muted PDZ domain—the D1 C-terminal processing protease (D1p) of is triggered by the presence of folding defects7,8. Although it is gen- the green alga Scenedesmus obliquus—involves a kinetic competition © 2010 Nature America, Inc. All rights reserved. All rights Inc. America, Nature © 2010 erally assumed that the exposure of aggregation-prone hydrophobic between folding to the native state and misfolding to a low-energy regions is the hallmark of misfolding9, only recently is it becoming off-pathway intermediate31. This type of misfolding is likely to be possible to outline the structural characteristics of folding defects that relatively rare in small protein domains, but it may be more common mediate the recognition of misfolded species by the quality control with larger proteins that have more complex topologies . We recently system10–12. Although major advances have been made in determin- engineered a circularly permuted variant of PTP-BL PDZ2, mimick- ing the structural features of on-pathway folding intermediates by ing the sequence connectivity of the naturally occurring circularly a variety of techniques, including NMR spectroscopy and Φ-value permuted PDZs. This variant is characterized by the presence of an analysis13–18, the primary aim of these studies has been to define the intermediate, which is stabilized by the circular permutation16,21. The rules that govern the folding process. Thus, despite their key role in PDZ family thus offers the possibility to investigate the determinants initiating pathological events, we are just beginning to clarify the of folding and misfolding in the context of divergent sequence and structural features of misfolded metastable states19,20. sequence connectivity, within the same three-dimensional frame. In this study we consider the problem of characterizing the struc- To map the structural features of both the productive and nonpro- ture of a misfolded intermediate and of defining the molecular events ductive folding pathways of this small protein domain, we carried out that determine whether nonproductive misfolding pathways are taken, a Φ-value analysis, whose results were then incorporated as structural rather than the correct folding pathway. In order to address these restraints in molecular dynamics simulations of D1pPDZ. Our results questions, we consider a PDZ domain protein that has been recently indicate that the structural features in the productive folding transi- shown to populate an off-pathway intermediate21. PDZ domains tion state of D1pPDZ resemble the early transition state for folding 1Istituto Pasteur–Fondazione Cenci Bolognetti and Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Università di Roma ‘La Sapienza’, Rome, Italy. 2Department of Chemistry, University of Cambridge, Cambridge, UK. 3Present address: Department of Human Genetics, Katholieke Universiteit Leuven, Herestraat, Leuven, Belgium. 4These authors contributed equally to this work. Correspondence should be addressed to M.V. ([email protected]). Received 31 March; accepted 8 October; published online 14 November 2010; doi:10.1038/nsmb.1956 NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 17 NUMBER 12 DECEMBER 2010 1431 ARTICLES 28 of the canonical PTP-BL PDZ2, which we described previously , a 8.0 b and thus suggest the existence of a conserved productive folding pathway, despite the low sequence identity and alternative sequence 7.5 connectivity . Our study, however, also reveals how specific events can cause the failure of this conserved folding mechanism. Our ana- 7.0 lysis of the nonproductive pathway indicates that the misfolded inter- mediate of D1pPDZ, despite having an overall native-like topology, is characterized by a non-native and nonfunctional arrangement of 6.5 the ligand-binding pocket region, caused by the incorrect docking of Fluorescence (relative units) 6.0 the N-terminal β-hairpin onto the remainder of the structure. Taken 0.01 0.1 1.0 10 0.01 0.1 1.0 together, our results indicate that the productive folding of D1pPDZ Time (s) Time (s) is driven by the formation of interactions in the main β-sheet and its docking with α2, whereas misfolding is caused by the incorrect docking of the N-terminal β-hairpin. RESULTS Φ-value analysis k k k k A general and challenging problem in studying the structure of interme- 1 3 1 3 I D N I D N diates in the folding process is that these reactions are generally highly k2 k4 k2 k4 cooperative, so that any other state populated during the process is extremely difficult to observe by standard methods of structural biol- Figure 1 Folding kinetics of D1pPDZ. (a) Representative refolding trace ogy1. A powerful strategy to address this problem, and to obtain struc- of D1pPDZ. Gray line is the best fit to a double exponential. (b) Trace of a tural information about intermediate and transition states, is to carry mutant (L245A) that populates almost exclusively the misfolded intermediate. out a systematic comparison of the folding and unfolding rate constants The gray line is the best fit to a single exponential. Although refolding of for a series of mutational variants, from which it is possible to map out D1pPDZ is biphasic, the mutant shows only the fast phase, reflecting the transition from the denatured state to the misfolded intermediate. the interaction patterns in the intermediate and transition states. In this approach, the so-called Φ-value analysis32, the strength of the contacts is measured by a parameter—the Φ value—that represents the change D1pPDZ is biphasic (Fig. 1a), both in the presence and absence of in stability of an intermediate (or of a transition state) relative to that stabilizing salt31. We have previously demonstrated that such a bipha- of the native state. Thus, Φ = 1 indicates that the site of mutation is sic refolding is caused by the kinetic partitioning between two folding fully structured in the intermediate (or in the transition state), whereas pathways, one leading to the more stable native state and the other to Φ = 0 indicates that such a site is essentially unstructured, and inter- the less stable off-pathway intermediate (Fig. 1a). In the present study, mediate values usually represent a partial degree of formation of the we carried out urea-induced kinetic (un)folding experiments in the native interactions in the intermediate (or in the transition state). presence of a stabilizing salt (0.4 M sodium sulfate). The majority of In this work, we used Φ-value analysis to study the properties of the D1pPDZ variants—30 mutants—had biphasic kinetic (un)folding the main states populated during the folding process of D1pPDZ. traces, similar to those observed for the wild type; the time courses Forty positions in the 95-residue sequence of this protein were probed were fitted to a double-exponential function, allowing unambiguous by conservative site-specific mutagenesis; mutants at such positions determination of all four microscopic rate constants.
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