An Oligomeric Equilibrium Intermediate as the Precursory Nucleus of Globular and Fibrillar Supramacromolecular Assemblies in a PDZ Domain

The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60-70°C small oligomers populate, showing a typical β-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of ∼12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of ∼50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60°C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ × mol⁻¹. Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.     

The Interconversion between a Flexible β-Sheet and a Fibril β-Arrangement Constitutes the Main Conformational Event during Misfolding of PSD95-PDZ3 Domain

The temperature-induced misfolding pathway of PDZ3, the third PDZ domain of the PSD95 neuronal protein, is populated by a trimeric β-sheet-rich intermediate state that leads to a stepwise and reversible formation of supramacromolecular structures. Using FTIR, we have found that misfolding of this pathway is not due to different ensembles of a variety of precursors, but comes mainly from the interconversion of a flexible β-sheet of the domain to wormlike fibrils. The appearance of the wormlike fibril FTIR component is also accompanied by a slight decrease of the band that corresponds to loops in the native state, whereas the rest of the regular elements of secondary structure are fairly well maintained upon misfolding. Transmission electron microscope micrographs have confirmed the presence of wormlike fibrils upon heating at 60°C, where the trimeric intermediate is maximally populated. Toxicity assays in the human neuroblastoma cell line SH-SY5Y show that cytotoxicity increases as the aggregation pathway proceeds. NMR analysis of chemical shifts as a function of temperature has revealed, as one of the main conformational aspects of such an interconversion at the residue level, that the β-sheet arrangement around strand β3 promotes the change that drives misfolding of the PDZ3 domain.     

Equilibrium unfolding of the PDZ domain of β2-syntrophin

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, T(m) and T(max) denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 and the results of molecular dynamics simulations indicate that this behavior of β2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.     

A Structural Portrait of the PDZ Domain Family

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family.     

Structural Basis for Asymmetric Association of the βPIX Coiled Coil and Shank PDZ

βPIX (p21-activated kinase interacting exchange factor) and Shank/ProSAP protein form a complex acting as a protein scaffold that integrates signaling pathways and regulates postsynaptic structure. Complex formation is mediated by the C-terminal PDZ binding motif of βPIX and the Shank PDZ domain. The coiled-coil (CC) domain upstream of the PDZ binding motif allows multimerization of βPIX, which is important for its physiological functions. We have solved the crystal structure of the βPIX CC-Shank PDZ complex and determined the stoichiometry of complex formation. The βPIX CC forms a 76-Å-long parallel CC trimer. Despite the fact that the βPIX CC exposes three PDZ binding motifs in the C-termini, the βPIX trimer associates with a single Shank PDZ. One of the C-terminal ends of the CC forms an extensive β-sheet interaction with the Shank PDZ, while the other two ends are not involved in ligand binding and form random coils. The two C-terminal ends of βPIX have significantly lower affinity than the first PDZ binding motif due to the steric hindrance in the C-terminal tails, which results in binding of a single PDZ domain to the βPIX trimer. The structure shows canonical class I PDZ binding with a β-sheet interaction extending to position − 6 of βPIX. The βB-βC loop of Shank PDZ undergoes a conformational change upon ligand binding to form the β-sheet interaction and to accommodate the bulky side chain of Trp − 5. This structural study provides a clear picture of the molecular recognition of the PDZ ligand and the asymmetric association of βPIX CC and Shank PDZ.     

The effect of pH on the structure, binding and model membrane lysis by cecropin B and analogs

Cecropins are a group of anti-bacterial, cationic peptides that have an amphipathic N-terminal segment, and a largely hydrophobic C-terminal segment and normally form a helix-hinge-helix structure. In this study, the ability of cecropin B (CB) and two analogs to lyse phospholipid bilayers, which have two levels of anionic content, has been examined by dye-leakage measurements over the pH range 2. 0-12.0. The two analogs differ from the natural peptide by having either two amphipathic segments (CB1) or two hydrophobic segments (CB3). All these peptides (except CB3 on low anionic content bilayers where it is not active) have maximal lytic activity on both types of bilayers at high pH. However, the pattern of secondary structure formation on these bilayers by the peptides, as measured by circular dichroism (CD), and the pattern of their ability to bind lipid monolayers, as measured using a biosensor, do not directly correlate with the pattern of their lytic ability. CB and CB1 with low anionic content bilayers have secondary structures as measured by CD with a similar pattern to membrane lysis, but binding is maximal near neutral, not high, pH. CB3 has some secondary structures on low anionic content bilayers at low pH and this becomes maximal over the basic range, but CB3 neither binds to nor lyses with these lipid layers. On high anionic content lipid layers, all peptides show high levels of secondary structures over most of the pH range and maximal binding at neutral pH (except for CB3, which does not bind). All three peptides lyse with high anionic content bilayers, but show no activity at neutral pH and reach maximal activity at very high pH. This work shows that pH is a major factor in the capability of antibacterial peptides to lyse with liposomes and that secondary structure and binding ability may not be the main determinants.     

Two conserved residues govern the salt and pH dependencies of the binding reaction of a PDZ domain

PDZ domains are protein-protein interaction modules found in hundreds of human proteins. Their binding reactions are sensitive to variations in salt and pH but the basis of the respective dependence has not been clear. We investigated the binding reaction between PSD-95 PDZ3 and a peptide corresponding to a native ligand with protein engineering in conjunction with stopped-flow and equilibrium fluorimetry and found that the two conserved residues Arg-318 and His-372 were responsible for the salt and pH dependencies, respectively. The basis of the salt-dependent variation of the affinity was explored by mutating all charged residues in and around the peptide-binding pocket. Arg-318 was found to be crucial, as mutation to alanine obliterated the effect of chloride on the binding constants. The direct interaction of chloride with Arg-318 was demonstrated by time-resolved urea denaturation experiments, where the Arg-318 --> Ala mutant was less stabilized by addition of chloride as compared with wild-type PDZ3. We also demonstrated that protonation of His-372 was responsible for the increase of the equilibrium dissociation constant at low pH. Both chloride concentration and pH (during ischemia) vary in the postsynaptic density, where PSD-95 is present, and the physiological buffer conditions may thus modulate the interaction between PSD-95 and its ligands through binding of chloride and protons to the "molecular switches" Arg-318 and His-372, respectively.     

Folding of pig gastric mucin non-glycosylated domains: a discrete molecular dynamics study

Mucin glycoproteins consist of tandem-repeating glycosylated regions flanked by non-repetitive protein domains with little glycosylation. These non-repetitive domains are involved in polymerization of mucin and play an important role in the pH-dependent gelation of gastric mucin, which is essential for protecting the stomach from autodigestion. We examine folding of the non-repetitive sequence of PGM-2X (242 amino acids) and the von Willebrand factor vWF-C1 domain (67 amino acids) at neutral and low pH using discrete molecular dynamics (DMD) in an implicit solvent combined with a four-bead peptide model. Using the same implicit solvent parameters, folding of both domains is simulated at neutral and low pH. In contrast to vWF-C1, PGM-2X folding is strongly affected by pH as indicated by changes in the contact order, radius of gyration, free-energy landscape, and the secondary structure. Whereas the free-energy landscape of vWF-C1 shows a single minimum at both neutral and low pH, the free-energy landscape of PGM-2X is characterized by multiple minima that are more numerous and shallower at low pH. Detailed structural analysis shows that PGM-2X partially unfolds at low pH. This partial unfolding is facilitated by the C-terminal region GLU236-PRO242, which loses contact with the rest of the domain due to effective “mean-field” repulsion among highly positively charged N- and C-terminal regions. Consequently, at low pH, hydrophobic amino acids are more exposed to the solvent. In vWF-C1, low pH induces some structural changes, including an increased exposure of CYS at position 67, but these changes are small compared to those found in PGM-2X. For PGM-2X, the DMD-derived average β-strand propensity increases from 0.26 ± 0.01 at neutral pH to 0.38 ± 0.01 at low pH. For vWF-C1, the DMD-derived average β-strand propensity is 0.32 ± 0.02 at neutral pH and 0.35 ± 0.02 at low pH. The DMD-derived structural information provides insight into pH-induced changes in the folding of two distinct mucin domains and suggests plausible mechanisms of the aggregation/gelation of mucin.     

The structural flexibility of the shank1 PDZ domain is important for its binding to different ligands

The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3 Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain.     

Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure

Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-B_N forms a coiled coil structure at acidic pH. The protonation of a residue with pK = 4.8 ± 0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-B_N, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-B_N overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide (⁶⁰W-E⁸⁵) designed from the sequence of the amino acid stretch of SP-B_N predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s_{20, w} = 0.55 S; Mr ~ 3 kDa) and peptide association (s_{20, w} = 1.735 S; Mr = ~ 14 kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N = 6 and M = 4 (Mr = 14692 Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular.     

N-Terminal domain of Bombyx mori fibroin mediates the assembly of silk in response to pH decrease

Fibroins serve as the major building blocks of silk fiber. As the major component of fibroin, the fibroin heavy chain is a considerably large protein comprising N-terminal and C-terminal hydrophilic domains and 12 highly repetitive Gly-Ala-rich regions flanked by internal hydrophilic blocks. Here, we show the crystal structure of the fibroin N-terminal domain (FibNT) at pH?4.7, revealing a remarkable double-layered anti-parallel β-sheet with each layer comprising two FibNT molecules entangled together. We also show that FibNT undergoes a pH-responsive conformational transition from random coil to β-sheets at around pH?6.0. Dynamic light scattering demonstrates that FibNT tends to oligomerize as pH decreases to 6.0, and electron microscopy reveals micelle-like oligomers. Our results are consistent with the micelle assembly model of silk fibroin and, more importantly, show that the N-terminal domain in itself has the capacity to form micelle-like structures in response to pH decrease. Structural and mutagenesis analyses further reveal the important role of conserved acidic residues clustered in FibNT, such as Glu56 and Asp100, in preventing premature β-sheet formation at neutral pH. Collectively, we suggest that FibNT functions as a pH-responsive self-assembly module that could prevent premature β-sheet formation at neutral pH yet could initiate fibroin assembly as pH decreases along the lumen of the posterior silk gland to the anterior silk gland.     

Psychosine remodels model lipid membranes at neutral pH

β-Galactosylsphingosine or psychosine (PSY) is a single chain sphingolipid with a cationic group, which is degraded in the lysosome lumen by β-galactosylceramidase during sphingolipid biosynthesis. A deficiency of this enzyme activity results in Krabbe's disease and PSY accumulation. This favors its escape to extralysosomal spaces, with its pH changing from acidic to neutral. We studied the interaction of PSY with model lipid membranes in neutral conditions, using phospholipid vesicles and monolayers as classical model systems, as well as a complex lipid mixture that mimics the lipid composition of myelin. At pH 7.4, when PSY is mainly neutral, it showed high surface activity, self-organizing into large structures, probably lamellar in nature, with a CMC of 38 ± 3 μM. When integrated into phospholipid membranes, PSY showed preferential partition into disordered phases, shifting phase equilibrium. The presence of PSY reduces the compactness of the membrane, making it more easily compressible. It also induces lipid domain disruption in vesicles composed of the main myelin lipids. The surface electrostatics of lipid membranes was altered by PSY in a complex manner. A shift to positive zeta potential values evidenced its presence in the vesicles. Furthermore, the increase of surface potential and surface water structuring observed may be a consequence of its location at the interface of the positively charged layer. As Krabbe's disease is a demyelinating process, PSY alteration of the membrane phase state, lateral lipid distribution and surface electrostatics appears important to the understanding of myelin destabilization at the supramolecular level.     

Trifluoroethanol-induced unfolding of concanavalin A: equilibrium and time-resolved optical spectroscopic studies

Conformational structure changes in concanavalin A (Con A), a legume lectin protein which is composed of 18 beta-strands, induced by dissolving in 50% trifluoroethanol (TFE) were monitored at neutral and low pH by far- and near-UV circular dichroism (CD), fluorescence, and FTIR under equilibrium conditions. Stopped-flow studies using CD and fluorescence as well as FTIR, at low and high protein concentration, respectively, were carried out to follow the time-dependent conformation changes occurring after rapid mixing of the protein with TFE. Equilibrium CD results show that, upon addition of TFE, low-concentration Con A transforms to a highly alpha-helical conformation at both neutral and low pH. However, at neutral pH under high protein concentration conditions, aggregation and precipitation are eventually detected with FTIR, indicating that a final beta-structure is attained. Stopped-flow fluorescence shows the existence of an unfolding intermediate for pH 2.0 and 4.5, which could be related to the dissociation of the dimer form. However, evidence for an intermediate is not obtained at pH 6.7, where the native protein is a tetramer. Stopped-flow FTIR is consistent with these results, indicating formation of a H(+)-stabilized intermediate alpha-helical conformation before aggregation develops. Con A in TFE provides an example of an intermediate with non-native secondary structure appearing on the unfolding pathway. On the basis of the kinetic results obtained, an unfolding mechanism is proposed and some stable intermediates are identified. In turn, the slow structural change of Con A induced by TFE provides a useful model system for study of protein unfolding due to its accessibility with several spectroscopic and kinetic tools.     

Structure of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.     

The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

Structure, dynamics and binding characteristics of the second PDZ domain of PTP-BL

The PDZ domains of the protein tyrosine phosphatase PTP-BL mediate interactions by binding to specific amino acid sequences in target proteins. The solution structure of the second PDZ domain of PTP-BL, PDZ2, displays a compact fold with six β strands and two α-helices. A unique feature of this domain compared to the canonical PDZ fold is an extended flexible loop at the base of the binding pocket, termed L1, that folds back onto the protein backbone, a feature that is shared by both the murine and human orthologues. The structure of PDZ2 differs significantly from the orthologous human structure. A comparison of structural quality indicators clearly demonstrates that the PDZ2 ensemble is statistically more reasonable than that of the human orthologue. The analysis of ¹⁵N relaxation data for PDZ2 shows a normal pattern, with more rigid secondary structures and more flexible loop structures. Close to the binding pocket, Leu85 and Thr88 display greater mobility when compared to surrounding residues. Peptide binding studies demonstrated a lack of interaction between murine PDZ2 and the C terminus of the murine Fas/CD95 receptor, suggesting that the Fas/CD95 receptor is not an in vivo target for PDZ2. In addition, PDZ2 specifically binds the C termini of both human Fas/CD95 receptor and the RIL protein, despite RIL containing a non-canonical PDZ-interacting sequence of E-x-V. A model of PDZ2 with the RIL peptide reveals that the PDZ2 binding pocket is able to accommodate the bulkier side-chain of glutamic acid while maintaining crucial protein to peptide hydrogen bond interactions.     

Molecular dynamics simulations to investigate the relationship between the structural stability and amyloidogenesis of the wild-type and N-terminal hexapeptide deletion ΔN6 β2-microglobulin

β2-Microglobulin (β2m) forms amyloid fibrils in patients undergoing long-term haemodialysis, leading to dialysis-related amyloidosis. Proteolysis of the N-terminal region of β2m results in a truncation of the six N-terminal residues (ΔN6 β2m) in ～30% of the β2m molecules extracted from ex vivo fibrils. The ΔN6 β2m has been shown to exhibit a higher tendency for self-association comparing to the wild-type (wt) β2m, particularly at neutral pH. In order to gain atomic insights into the early stages of amyloid formation of the wt and ΔN6 β2m, various molecular dynamics simulations were conducted to investigate the stability and dynamics of these two molecules at various temperatures and neutral pH in this study. Our results, in agreement with previous experimental results, indicate that the structural stability of the ΔN6 β2m is lower than that of the wt β2m. It can be attributed to fact that the removal of the N-terminal six residues results in the loss of the salt–bridge interaction between residues R3 and D59, leading to the increased solvent exposure of the K3 peptide. It further allows water molecules to destabilise the interior region of the K3 peptide, leading to the elongation between the B- and E-strands. It may further accelerate the conformational changes of the ΔN6 β2m, leading to the formation of amyloid fibrils more readily at neutral pH. Our results also suggest that the K3 peptide may be a potential initiation site of amyloid formation for the ΔN6 β2m due to its increased solvent exposure. We further suggest that fibril morphology of the ΔN6 β2m formed at neutral pH is similar to that of the wt β2m formed at low pH (1.5–3) since they adopt the similar conformation with the elongation between B- and E-strands for their partially unfolded amyloidogenic intermediates.     

Structural determinants governing β-arrestin2 interaction with PDZ proteins and recruitment to CRFR1

β-Arrestins are multifunctional adaptor proteins best know for their vital role in regulating G protein coupled receptor (GPCR) trafficking and signaling. β-arrestin2 recruitment and receptor internalization of corticotropin-releasing factor receptor 1 (CRFR1), a GPCR whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects, have previously been shown to be modulated by PDZ proteins. Thus, a structural characterization of the interaction between β-arrestins and PDZ proteins can delineate potential mechanism of PDZ-dependent regulation of GPCR trafficking. Here, we find that the PDZ proteins PSD-95, MAGI1, and PDZK1 interact with β-arrestin2 in a PDZ domain-dependent manner. Further investigation of such interaction using mutational analyses revealed that mutating the alanine residue at 175 residue of β-arrestin2 to phenylalanine impairs interaction with PSD-95. Additionally, A175F mutant of β-arrestin2 shows decreased CRF-stimulated recruitment to CRFR1 and reduced receptor internalization. Thus, our findings show that the interaction between β-arrestins and PDZ proteins is key for CRFR1 trafficking and may be targeted to mitigate impaired CRFR1 signaling in mental and psychiatric disorders.