Understanding the molecular basis of folding cooperativity through a comparative analysis of a multidomain protein and its isolated domains

Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multi-domain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multi-domain protein may either fold independently on each other or exhibit inter-dependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that, despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single co-operative unit. By exploiting quantitatively the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short auto-inhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multi-domain systems

Folding and Binding Mechanisms of the SH2 Domain from Crkl

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains

Indagini preliminari e caratterizzazione di siti contaminati e discariche abusive. Impatti sulla SSL

I lavoratori coinvolti nella gestione operativa di siti contaminati e di discariche abusive, sono potenzialmente esposti a rischi specifici connessi alle caratteristiche di tali particolari area di lavoro, aggiuntivi rispetto a quelli che tipicamente sono riscontrabili in caso di realizzazione di opere civili e/o edili. Nel caso di siti contaminati ci si riferisce in particolare ai rischi chimico e biologico, mentre riguardo le discariche abusive, o i depositi incontrollati di rifiuti, anche a quelli connessi a specifiche condizioni pericolose, quali la presenza di rifiuti infiammabili, l’instabilità geotecnica, la presenza di biogas e la presenza di percolato. È evidente che nelle fasi iniziali, come i sopralluoghi conoscitivi, le indagini preliminari e le attività di caratterizzazione, la tipologia, l’entità e l’estensione della contaminazione delle matrici ambientali (suolo, sottosuolo, acque superficiali e profonde), come anche le caratteristiche chimiche, fisiche e meccaniche dei rifiuti di discarica, non sono note. Nonostante ciò, ai sensi sia del d.lgs. 81/2008 che del d.lgs. 152/2006 (Allegato 3, Parte Quarta, Titolo V), per la tutela dei lavoratori, la gestione di tutti i rischi potenzialmente presenti deve sempre essere garantita. Dall’esperienza maturata nel settore, in Italia si riscontra una sostanziale carenza di indirizzi, criteri e strumenti operativi a tale riguardo. Pertanto l’Inail (Istituto nazionale italiano per l’assicurazione contro gli infortuni sul lavoro) ha avviato un progetto di ricerca finalizzato a supportare la risoluzione di tali criticità. Nel presente contributo vengono illustrati criteri utili per una adeguata valutazione e gestione dei rischi specifici di settore ai quali i lavoratori possono essere esposti nelle fasi di sopralluogo preliminare e caratterizzazione di siti contaminati e di discariche abusive. Per quest’ultime un particolare approfondimento è rivolto alle indagini di tipo indiretto, ancora poco usate in fase di indagine preliminare, nonostante costituiscano strumenti per la prevenzione di eventi dannosi, generalmente poco prevedibili

Colangiografia intraoperatoria in corso di colecistectomia laparoscopica: selettiva o di routine?

Il ruolo della colangiografia intraoperatoria (CIO) risultò contro - verso già dalla sua introduzione ad opera di Mirizzi nel 1931 e divenne argomento sempre più discusso dopo l’avvento della colecistec - tomia laparoscopica (VLC) nel 1988. Scopo del presente lavoro è quel - lo di definire, sulla base di un’analisi retrospettiva di una casistica di CIO eseguite selettivamente, il ruolo della CIO in corso di VLC. Dal mese di dicembre 1991 al mese di giugno 2001 sono stati ricoverati 597 pazienti di cui 552 in elezione e 45 in urgenza. Di 552 pazienti, 62 presentavano almeno un criterio di sospetto per litia - si della via biliare principale (VBP) e venivano trattati con ERCP completata con una sfinterotomia endoscopica (ES) se concomitava litiasi della VBP, mentre nei restanti 490 si eseguiva una VLC; s o n o state effettuate in totale 10 CIO, di cui 2 nel gruppo trattato con ERCP ed 8 nel gruppo delle VLC. I 145 pazienti ricoverati in urgen - za sono stati sottoposti a VLC; in 43 casi si è reso necessario eseguire la CIO. Sul totale dei pazienti, 2 sono stati i casi di lesione iatrogenica della VBP (0,33%) ed in entrambi i casi non è stata effettuata la CIO. Quest’ultima ha determinato un tempo aggiuntivo medio di 27 m i n . Sulla base dell’esperienza degli Autori e della letteratura si può affermare che l’uso routinario della CIO non sembra efficace nella ridu - zione delle lesioni iatrogeniche della VBP, mentre può aggravare pesan - temente i costi dell’intervento chirurgico allungandone i tempi. Sembra invece efficace nello svelare una calcolosi della VBP non clinicamente evidente, ma attualmente non è ancora chiaro l’esatto vantaggio che questa acquisizione ha nella pratica clinica. Gli Autori concludono affermando che ulteriori studi su base prospettico-randomizzata sono necessari per chiarire l’esatto ruolo della CIO nei confronti della VL

SH2 Domains: Folding, Binding and Therapeutical Approaches

SH2 (Src Homology 2) domains are among the best characterized and most studied protein-protein interaction (PPIs) modules able to bind and recognize sequences presenting a phosphorylated tyrosine. This post-translational modification is a key regulator of a plethora of physiological and molecular pathways in the eukaryotic cell, so SH2 domains possess a fundamental role in cell signaling. Consequently, several pathologies arise from the dysregulation of such SH2-domains mediated PPIs. In this review, we recapitulate the current knowledge about the structural, folding stability, and binding properties of SH2 domains and their roles in molecular pathways and pathogenesis. Moreover, we focus attention on the different strategies employed to modulate/inhibit SH2 domains binding. Altogether, the information gathered points to evidence that pharmacological interest in SH2 domains is highly strategic to developing new therapeutics. Moreover, a deeper understanding of the molecular determinants of the thermodynamic stability as well as of the binding properties of SH2 domains appears to be fundamental in order to improve the possibility of preventing their dysregulated interactions

Exploring the effect of tethered domains on the folding of Grb2 protein

Two thirds of eukaryotic proteins have evolved as multidomain constructs, and in vivo, domains fold within a polypeptide chain, with inter-domain interactions possibly crucial for correct folding. However, to date, most of the experimental folding studies are based on domains in isolation. In an effort to better understand multidomain folding, in this work we analyzed, through equilibrium and kinetic folding experiments, the folding properties of the Growth factor receptor-bound protein 2 (Grb2), composed by one SRC homology 2 domain flanked by two SRC homology 3 domains. In particular we compared the kinetic features of the multidomain construct with the domains expressed in isolation. By performing single and double mixing folding experiments, we demonstrated that the folding of the SH2 domain is kinetically trapped in a misfolded intermediate when tethered to the C-SH3. Importantly, within the multidomain construct, misfolding occurred independently if refolding is started with C-SH3 in its unfolded or native state. Interestingly, our data reported a peculiar scenario, in which SH2 and C-SH3 domain reciprocally and transiently interact during folding. Altogether, the analysis of kinetic folding data provided a quantitative description of the multidomain folding of Grb2 protein, discussed under the light of previous works on multidomain folding

Characterization of the folding and binding properties of the PTB domain of FRS2 with phosphorylated and unphosphorylated ligands

PTB (PhosphoTyrosine Binding) domains are protein domains that exert their function by binding phosphotyrosine residues on other proteins. They are commonly found in a variety of signaling proteins and are important for mediating protein-protein interactions in numerous cellular processes. PTB domains can also exhibit binding to unphosphorylated ligands, suggesting that they have additional binding specificities beyond phosphotyrosine recognition. Structural studies have reported that the PTB domain from FRS2 possesses this peculiar feature, allowing it to interact with both phosphorylated and unphosphorylated ligands, such as TrkB and FGFR1, through different topologies and orientations. In an effort to elucidate the dynamic and functional properties of these protein-protein interactions, we provide a complete characterization of the folding mechanism of the PTB domain of FRS2 and the binding process to peptides mimicking specific regions of TrkB and FGFR1. By analyzing the equilibrium and kinetics of PTB folding, we propose a mechanism implying the presence of an intermediate along the folding pathway. Kinetic binding experiments performed at different ionic strengths highlighted the electrostatic nature of the interaction with both peptides. The specific role of single amino acids in early and late events of binding was pinpointed by site-directed mutagenesis. These results are discussed in light of previous experimental works on these protein systems

Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners

SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein–protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain