Catalysis of proline isomerization and molecular chaperone activity in a tug-of-war

Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by peptidylprolyl isomerases, a superfamily of molecular chaperones. Here, we provide atomic insight into a tug-of-war between cis/trans isomerization and molecular chaperone activity. Catalysis of proline isomerization by cyclophilin A lowers the energy barrier for \u3b1-synuclein misfolding, while isomerase-binding to a separate, disease-associated protein region opposes aggregation. We further show that cis/trans isomerization outpowers the holding activity of cyclophilin A. Removal of the proline isomerization barrier through posttranslational truncation of \u3b1-synuclein reverses the action of the proline isomerase and turns it into a potent molecular chaperone that inhibits protein misfolding. The data reveal a conserved mechanism of dual functionality in cis/trans isomerases and define its molecular determinants acting on intrinsically disordered proteins

Role of myristoylation in modulating PCaP1 interaction with calmodulin

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca2+) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca2+/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca2+-CaM interaction and the functional impact of myristoylation in the complex formation and Ca2+ sensitivity of CaM remained to be elucidated. Herein, we investigated the direct interaction between Arabidopsis PCaP1 (AtPCaP1) and CaM1 (AtCaM1) using both myristoylated and non-myristoylated peptides corresponding to the N-terminal region of AtPCaP1. ITC analysis showed that AtCaM1 forms a high affinity 1:1 complex with AtPCaP1 peptides and the interaction is strictly Ca2+-dependent. Spectroscopic and kinetic Ca2+ binding studies showed that the myristoylated peptide dramatically increased the Ca2+-binding affinity of AtCaM1 and slowed the Ca2+ dissociation rates from both the C- and N-lobes, thus suggesting that the myristoylation modulates the mechanism of AtPCaP1 recognition by AtCaM1. Furthermore, NMR and CD spectroscopy revealed that the structure of both the N- and C-lobes of Ca2+-AtCaM1 changes markedly in the presence of the myristoylated AtPCaP1 peptide, which assumes a helical structure in the final complex. Overall, our results indicate that AtPCaP1 biological function is strictly related to the presence of multiple ligands, i.e., the myristoyl moiety, Ca2+ ions and AtCaM1 and only a full characterization of their equilibria will allow for a complete molecular understanding of the putative role of PCaP1 as signal protein

NMR Interaction studies of Human Liver Fatty Acid Binding Protein with putative ligands and associated proteins

Le molecole lipidiche, come acidi grassi, eicosanoidi e acidi biliari (BAs) sono essenziali per la sopravvivenza cellulare, in quanto possono fungere da fonti di energia, da substrati per la formazione di membrane oppure da molecole segnale per la regolazione del metabolismo cellulare. A causa della loro scarsa solubilit\ue0 e della loro potenziale citotossicit\ue0 necessitano di chaperon intracellulari che, legandole, aumentano la loro solubilit\ue0 in solventi acquosi. Le proteine che legano gli acidi grassi, FABPs (Fatty Acid Binding Proteins), appartengono alla famiglia delle iLBPs (intracellular lipid binding proteins), una classe di piccole proteine citoplasmatiche (di circa 14-15KDa) evolutivamente correlate fra loro, probabilmente implicate nel trasporto lipidico trans-cellulare. Le iLBP sono proteine versatili che partecipano ai processi nucleari e al mantenimento dell\u2019omeostasi e del metabolismo lipidico; per questo motivo sono state scelte come bersagli di farmaci contro lo sviluppo di dislipidemie. La FABP di fegato (LFABP), appartenente alla sotto-famiglia II delle FABP, \ue8 forse la pi\uf9 peculiare; a differenza degli altri membri pu\uf2 infatti accomodare, nella sua cavit\ue0 idrofobica, due molecole di acidi grassi a lunga catena, ma anche una grande variet\ue0 di molecole idrofobiche come gli esteri dell\u2019acil-CoA, fosfolipidi e acidi biliari. Per le sue particolari caratteristiche e l'alta concentrazione che pu\uf2 raggiungere all'interno degli epatociti (1-5% delle proteine solubili totali), si \ue8 ipotizzato che la LFABP umana (HLFABP) sia implicata nello sviluppo dei parassiti malarici, durante la fase epatica della malattia. Questa fase \ue8 asintomatica e potrebbe fornire nuove strategie per arrestare l'infezione. UIS3 \ue8 una piccola proteina trans-membrana, presumibilmente localizzata nella membrana vacuolare parassitofora (PVM), specificamente espressa negli sporozoiti infettivi ed essenziale per il loro sviluppo nella fase iniziale della malaria all\u2019interno del fegato. Un saggio di tipo yeast two hybrid, basato sulla proteina UIS3 derivante dal parassita malarico dei roditori P. yoelii (Py-UIS3), ha permesso di identificare LFABP come possibile partner di interazione. Inoltre, in un lavoro del 2008 di Ashwani e collaboratori, venne riportata un\u2019interazione diretta fra HLFABP e il dominio solubile di UIS3 di Plasmodium falciparum ( PfUIS3(130-229)). Al fine di ottenere informazioni vincolanti ad un livello atomico di risoluzione, l'interazione tra HLFABP e PfUIS3(130-229) \ue8 stata analizzata in dettaglio tramite spettroscopia di Risonanza Magnetica Nucleare(NMR). Inoltre, anche l'associazione con fosfolipidi e acidi grassi \ue8 stata analizzata attraverso NMR. I nostri dati, tuttavia, non hanno evidenziato alcuna interazione di PfUIS3(130-229) con HLFABP e/o molecole lipidiche, indicando la necessit\ue0 di ridefinire il modello attuale di importo di lipidi nei parassiti malarici mediato da LFABP. Nella seconda parte di questo progetto di ricerca \ue8 stata analizzata in dettaglio l\u2019interazione fra HLFABP e gli acidi grassi. L\u2019NMR e la spettroscopia di massa (MS) sono state utilizzate per la prima volta insieme per caratterizzare questa associazione. Campioni di HLFABP in complesso con oleato (OA) e palmitato (PA) sono stati preparati in acqua e successivamente analizzati utilizzando la tecnologia ESI-MS (Electron Spray Ionization Mass Spectroscopy) per determinarne la specificit\ue0, la stechiometria e l'affinit\ue0 relativa. I nostri dati sono concordi con la presenza di due siti di legame distinti con una diversa affinit\ue0 per gli acidi grassi. Sono poi stati allestiti degli esperimenti di competizione, titolando la proteina sia con OA che con PA; i campioni sono stati analizzati tramite ESI-MS ed i dati indicano che OA e PA competono effettivamente per lo stesso sito di associazione all'interno della proteina e che HLFABP ha una maggiore affinit\ue0 per gli acidi grassi instauri. Successivamente abbiamo sfruttato la potenza delle titolazioni 13C-NMR per indagare sia l'interazione fra HLFABP e acidi grassi marcati in 13C, che lo stato di ionizzazione dei ligandi legati nella tasca idrofobica della proteina. A livello globale, abbiamo sviluppato un metodo adatto per lo studio di altri membri della famiglia FABP, che, nonostante le loro dimensioni molto favorevoli, sono sistemi complessi per essere caratterizzati da una singola tecnica biofisica. Inoltre, questo metodo potr\ue0 essere applicato anche per lo studio di HLFABP in complesso con altri ligandi idrofobici. Nell'ultima parte di questo lavoro ci siamo concentrati sull'interazione tra HLFABP e gli acidi biliari, molecole anfipatiche, che facilitano l'assorbimento dei lipidi, del colesterolo e di vitamine liposolubili nell'intestino tenue. Gli acidi biliari subiscono un riciclaggio tra l'intestino ed il fegato, chiamato "Circolazione enteroepatica", che consente il recupero di quasi il 95% di queste preziose molecole. Dal momento che un trasportatore di acidi biliari non \ue8 stato ancora stato identificato negli epatociti dei mammiferi, abbiamo esplorato l\u2019associazione tra HLFABP e gli acidi biliari utilizzando una vasta gamma di tecniche biofisiche, coinvolgendo l'NMR, la spettroscopia di fluorescenza e la spettrometria di massa. L'interazione tra HLFABP e l\u2019acido glicocolico (GCA), il sale biliare pi\uf9 abbondante presente nel fegato umano, \ue8 stato ampiamente esplorato mediante NMR. L\u2019NMR \ue8 una fra le spettroscopie pi\uf9 potenti e versatili per l'analisi molecolare, poich\ue9 permette di caratterizzare la struttura delle macromolecole biologiche ed i loro complessi ad un livello atomico di risoluzione. Inoltre, l'NMR fornisce informazioni sulla dinamica proteica su una vasta gamma di scale dei tempi . Le dinamiche possono influenzare la velocit\ue0 e la via di ripiegamento delle proteine, cos\uec come l\u2019aggregazione, la catalisi e l\u2019associazione ad un ligando attraverso l\u2019adattamento indotto o la selezione conformazionale. Cos\uec, la determinazione delle dinamiche proteiche in soluzione \ue8 importante per comprendere l'intero spettro di funzioni macromolecolari svolte dalle proteine. Esperimenti di titolazione NMR ed esperimenti omonucleari 1H-1H NOESY, eseguiti con diversi schemi di marcatura isotopica, suggeriscono che HLFABP \ue8 in grado di legare una sola molecola di GCA. Inoltre, per complementare i dati NMR, \ue8 stata eseguita un\u2019analisi computazionale per calcolare la struttura del complesso, utilizzando il programma di docking HADDOCK. Successivamente, per meglio definire il legame, sono stati acquisiti esperimenti di rilassamento 15N sul backbone di HLFABP nella sua forma apo ed in complesso sia con GCA che con OA e sono state ottenute dinamiche residuo-specifiche su una vasta scala di tempi, che va dai ns ai ms. I nostri dati indicano chiaramente chele dinamiche veloci (ps-ns) non vengono influenzate particolarmente dal binding, mentre le dinamiche lente (\u3bcs-ms) sono mantenute o accentuate dopo il legame. Infine sono stati eseguiti esperimenti di scambio idrogeno/deuterio e CLEANEX per monitorare le zone pi\uf9 protette della proteina dallo scambio col solvente in presenza ed in assenza dei ligandi. In presenza di GCA \ue8 stato poi osservato un aumento della stabilit\ue0 proteica. La spettroscopia di fluorescenza e l'NMR sono state anche utilizzate per caratterizzare l'interazione fra HLFABP e un pool di acidi biliari con diverse modalit\ue0 di coniugazione e idrossilazione. I dati NMR mostrano che HLFABP pu\uf2 interagire con un\u2019ampia gamma di acidi biliari in modo complesso, attraverso la formazione di almeno uno stato attivato. In aggiunta, i nostri dati NMR suggeriscono che la struttura della proteina sia preformata per il legame delle diverse molecole idrofobiche. Infatti la rete di legami idrogeno non viene perturbata in modo significativo dall'aggiunta dei vari ligandi. Attraverso la spettroscopia NMR abbiamo poi dimostrato che HLFABP esiste come un insieme di conformeri in scambio veloce fra loro su una scala di tempi NMR. La spettroscopia di fluorescenza \ue8 stata impiegata per calcolare l\u2019affinit\ue0 di HLFABP verso i vari acidi biliari utilizzati nello screening e attraverso un saggio di competizione, utilizzando il DAUDA come composto fluorescente, \ue8 stata calcolata un\u2019affinit\ue0 \u3bcM (da 0.6-7.5 \u3bcM) in accordo con quella calcolata tramite spettroscopia NMR. L\u2019affinit\ue0 maggiore \ue8 stata ottenuta per quegli acidi biliari con caratteristiche di idrofobicit\ue0 maggiori. Infine, la presenza di complessi eterotipici, formati da HLFABP in complesso sia con acidi biliari che con acidi grassi, \ue8 stata analizzata tramite NMR e ESI-MS.Lipidic molecules such as fatty acids (FAs), eicosanoids and bile salts (BAs) are essential for cell survival because they serve as metabolic energy sources, substrates for membranes and signaling molecules for metabolic regulation. Due to their low solubility and in some case cytotoxicity they necessitate intracellular chaperons, which bind them, thus increasing their aqueous solubility. Fatty acid binding proteins (FABPs), belong to the Intracellular lipid binding proteins (iLBPs) family, a class of evolutionarily related small (14-15 KDa) cytoplasmic proteins, which have been proposed to be implicated in the transcellular transport of lipophilic ligands. Due to their participation in nuclear processes and lipid metabolism and homeostasis, they have recently been proposed as drug targets against the development of lipid related disorders. Among the other family members, liver fatty acid binding protein (LFABP), belonging to subfamily II of FABPs, is the most unique. Differently from the other FABPs, LFABP is able to accommodate two long chain FAs (LCFAs) molecules, but also a wide range of hydrophobic ligands, such as BAs, eicosanoids, Acyl-CoA esters and phospholipids. Due to its peculiar characteristics and the high concentration that it could reach within the hepatocytes (1-5% of total soluble proteins), human LFABP (HLFABP) has been hypothesized to be implicated in malaria parasites development, during the hepatic stage of the disease. The hepatic stage is asymptomatic and it would provide novel strategies for arresting the infection. UIS3 is a small transmembrane protein, presumably localized to the parasitophorous vacuolar membrane (PVM), specifically expressed in infective sporozoites, and essential for early-stage liver development. A yeast two-hybrid screen based on UIS3 of the rodent malaria parasite P. yoelii (Py-UIS3) identified mouse LFABP as an interacting host protein. In addition, a work of 2008 of Ashwani and collaborators reports a direct interaction between HLFABP and the soluble domain of UIS3 from Plasmodium falciparum (PfUIS3(130-229)). In order to gain binding information at an atomic level of resolution, the interaction between HLFABP and PfUIS3(130-229) was analyzed in detail exploiting Nuclear Mgnetic Resonance (NMR) spectroscopy. Furthermore, the direct binding of phospholipids and FAs to UIS3 was also analyzed by NMR. However, our data did not show any interaction of PfUIS3(130-229) with HLFABP and lipid molecules, calling for a redefinition of the current model of FABP-mediated lipid import by human malaria parasites. In the second part of this research project, we investigated in detail the interaction between HLFABP and fatty acids. For the first time NMR and MS spectroscopy were used in combination to characterize the binding between HLFABP and FAs. Samples of HLFABP in complex with palmitate (PA) or oleate (OA) were prepared in water and analyzed through Electron Spray Ionization mass spectroscopy (ESI-MS) to asses specificity, stoichiometry and relative affinity. Our data are in agreement with the presence of two distinct binding sites with different affinities for FAs. Competition experiments were also performed, titrating the protein with both PA and OA; OA and PA effectively compete for the same binding site within the protein binding pocket. Our results show that HLFABP has an higher affinity for unsaturated FAs. Successively, we exploited the power of 13C NMR titration data to investigate the interaction between HLFABP and 13C FAs and to get information about the ionization state of the bound ligands. Globally, we developed a method suitable for the study of other FABP family members, which, despite their favorable size are really challenging systems to be characterized by only a singular biophysical technique. In addition, this method could be also applied to the study of HLFABP in complex with other hydrophobic ligands. In the last part of this work we focused on the interaction between HLFABP and BAs, amphipathic molecules, which in the small intestine facilitate the absorption of dietary lipids, cholesterol, and fatsoluble vitamins. BAs undergo a recycling pathway between the intestine and the liver, called \u201centerohepatic circulation\u201d, which allows the recovery of almost the 95% of these precious molecules. Since a BA carrier within the hepatocytes has not been identified yet, we explored the interaction between HLFABP and BAs using a wide range of biophysical techniques, involving NMR, florescence and mass spectroscopy. The interaction between HLFABP and glycocholic acid (GCA), the most abundant bile salt present in human liver, was extensively explored using NMR spectroscopy technique. NMR is one of the most powerful and versatile spectroscopic technique for molecular analysis, since it allows to characterize biological macromolecules and their complexes at an atomic level of resolution. In addition, NMR provides information about protein dynamics on a wide range of time scales. Dynamics can affect the rate and pathway of protein folding, as well as misfolding and aggregation, catalysis and also binding via induced fit or conformational selection. Thus, the determination of protein dynamics in solution is important for realizing the full spectrum of macromolecular functions and for predicting and engineering protein behavior. NMR titration experiments and 1H-1H homonuclear NOESY filtered experiments, performed with different labeling schemes, suggested that HLFABP is able to accommodate only one molecule of GCA. To complement NMR data, a model of the complex was obtained through a computational analysis, using the docking program HADDOCK. To better characterize the binding, 15N backbone relaxation experiments on HLFABP in its apo form and in complex with either GCA or OA were recorded and residue specific dynamics, on a time scale ranging from ps to ms, were obtained. Fast time scale dynamics are not significantly perturbed upon OA/GCA addition, while slow motions are retained or enhanced upon binding. Hydrogen/deuterium exchange and CLEANEX experiments were also performed to get information on solvent accessibility to individual sites and to detect protein dynamics occurring on a much slower time scale. An increase in protein stability upon GCA/OA binding was observed. For the first time NMR and fluorescence spectroscopy were combined on a BA pool, with different pattern of conjugation and hydroxylation. The NMR data show that HLFABP can interact with a wide range of bile salts, through a complex pathway, involving at least one activated state. In addition the hydrogen bond network was not significantly perturbed upon ligand addition, indicating that the scaffold of the protein is preformed to bind such kind of ligands. Through NMR spectroscopy, we demonstrated also that HLFABP exists as an ensemble of conformers in fast exchange on an NMR time scale. Fluorescence spectroscopy was used to calculate the affinity of HLFABP toward the different BAs employed in the study. An affinity in the \ub5M range (spanning form 0.6-7.5\ub5M) was obtained through DAUDA displacement assay, in close agreement with the ones calculated by NMR. The higher affinity was obtained for those BAs displaying high hydrophobic properties. Finally we analyzed both by NMR and mass spectroscopy the existence of an heterotypic complex constituted by HLFABP in complex with both GCA and FAs, which is likely the conformation assumed by the protein in vivo

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca2+) binding site that is non-functional, thus displaying large variance in Ca2+ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca2+ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca2+ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins

Bile salt recognition by human liver fatty acid binding protein

Fatty acid binding proteins (FABPs) act as intracellular carriers of lipid molecules, and play a role in global metabolism regulation. Liver FABP (L-FABP) is prominent among FABPs for its wide ligand repertoire, which includes long-chain fatty acids as well as bile acids (BAs). In this work, we performed a detailed molecular- and atomic-level analysis of the interactions established by human L-FABP with nine BAs to understand the binding specificity for this important class of cholesterol-derived metabolites. Protein-ligand complex formation was monitored using heteronuclear NMR, steady-state fluorescence spectroscopy, and mass spectrometry. BAs were found to interact with L-FABP with dissociation constants in the narrow range of 0.6-7 \u3bcm; however, the diverse substitution patterns of the sterol nucleus and the presence of side-chain conjugation resulted in complexes endowed with various degrees of conformational heterogeneity. Trihydroxylated BAs formed monomeric complexes in which single ligand molecules occupied similar internal binding sites, based on chemical-shift perturbation data. Analysis of NMR line shapes upon progressive addition of taurocholate indicated that the binding mechanism departed from a simple binary association equilibrium, and instead involved intermediates along the binding path. The co-linear chemical shift behavior observed for L-FABP complexes with cholate derivatives added insight into conformational dynamics in the presence of ligands. The observed spectroscopic features of L-FABP/BA complexes, discussed in relation to ligand chemistry, suggest possible molecular determinants of recognition, with implications regarding intracellular BA transport. Our findings suggest that human L-FABP is a poorly selective, universal BA binder

Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level

Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell

The unique ligand binding features of subfamily-II iLBPs with respect to bile salts and related drugs

Intracellular lipid binding proteins (iLBPs) are a family of evolutionarily related small cytoplasmic proteins implicated in the transcellular transport of lipophilic ligands. Subfamily-II iLBPs include the liver fatty acid binding protein (L-FABP), and the ileal and the liver and ileal bile acid binding proteins (L-BABP and I-BABP). Atomic-level investigations during the past 15-20 years have delivered relevant information on bile acid binding by this protein group, revealing unique features including binding cooperativity, promiscuity, and site selectivity. Using NMR spectroscopy and other biophysical techniques, our laboratories have contributed to an understanding of the molecular determinants of some of these properties and their generality among proteins from different animal species. We focused especially on formation of heterotypic complexes, considering the mixed compositions of physiological bile acid pools. Experiments performed with synthetic bile acid derivatives showed that iLBPs could act as targets for cell-specific contrast agents and, more generally, as effective carriers of amphiphilic drugs. This review collects the major findings related to bile salt interactions with iLBPs aiming to provide keys for a deeper understanding of protein-mediated intracellular bile salt trafficking