Structures of single‐layer β‐sheet proteins evolved from β‐hairpin repeats

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.     

Re‐engineering a β‐lactamase using prototype peptides from a library of local structural motifs

B. licheniformis exo‐small β‐lactamase (ESBL) has a complex architecture with twelve α helices and a five‐stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL α helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.     

Selection and structural analysis of de novo proteins from an α3β3 genetic library

The construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.     

A dimer model of human calcitonin13‐32 forms an α‐helical structure and robustly aggregates in 50% aqueous 2,2,2‐trifluoroethanol solution

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13‐32 aggregated to a greater degree than native hCT under aqueous 2,2,2‐trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine‐T binding assays and atomic force microscopy suggest that the α‐helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β‐sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α‐helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Structural characterization of a neurotoxic threonine‐rich peptide corresponding to the human prion protein α2‐helical 180–195 segment,and comparison with full‐length α2‐helix‐derived peptides

The 173–195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ‘spots’ of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation‐prone conformations. Here, we report CD and NMR studies on the α2‐helix‐derived peptide of maximal length (hPrP[180–195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other α2‐helix‐derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C‐terminal sequence of the PrP^C full‐length α2‐helix and includes the highly conserved threonine‐rich 188–195 segment. At neutral pH, its conformation is dominated by β‐type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of α‐helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173–179 segment, as occurring in wild‐type and mutant peptides corresponding to the full‐length α2‐helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180–195 parental region in hPrP^C makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full‐length α2‐helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Basic conformers in β‐peptides

The conformation of oligomers of β‐amino acids of the general type Ac‐[β‐Xaa]_n‐NHMe (β‐Xaa = β‐Ala, β‐Aib, and β‐Abu; n = 1–4) was systematically examined at different levels of ab initio molecular orbital theory (HF/6‐31G*, HF/3‐21G). The solvent influence was considered employing two quantum‐mechanical self‐consistent reaction field models. The results show a wide variety of possibilities for the formation of characteristic elements of secondary structure in β‐peptides. Most of them can be derived from the monomer units of blocked β‐peptides with n = 1. The stability and geometries of the β‐peptide structures are considerably influenced by the side‐chain positions, by the configurations at the C^α‐ and C^β‐atoms of the β‐amino acid constituents, and especially by environmental effects. Structure peculiarities of β‐peptides, in particular those of various helix alternatives, are discussed in relation to typical elements of secondary structure in α‐peptides. © 1999 John Wiley & Sons, Inc. Biopoly 50: 167–184, 1999     

Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.     

β‐sheet assembly in amyloidogenic glutamic acid nanostructures: Insights from X‐ray scattering and infrared nanospectroscopy

Glutamic acid–rich peptides are crucial to a variety of biological processes, including glutamatergic neurotransmission and immunological defense. Glutamic acid sequences often exhibit unusual organization into β₂‐type sheets, where bifurcated H bonds formed between glutamic acid side chains and NH in amide bonds on adjacent β‐strands play a paramount role for stabilizing the molecular assembly. Herein, we investigate the self‐assembly and supramolecular structure of simplified models consisting of alternating glutamic acid/phenylalanine residues. Small‐angle X‐ray scattering and atomic force microscopy show that the aggregation pathway is characterized by the formation of small oligomers, followed by coalescence into nanofibrils and nanotapes. Amyloidogenic features are further demonstrated through fiber X‐ray diffraction, which reveal molecular packing according to cross‐β patterns, where β‐strands appear perpendicularly oriented to the long axis of nanofibrils and nanotapes. Nanoscale infrared spectroscopy from individual nanoparticles on dried samples shows a remarkable decrease of β₂‐sheet content, accompanied by growth of standard β‐sheet fractions, indicating a β₂‐to‐β₁ transition as a consequence of the release of solvent from the interstices of peptide assemblies. Our findings highlight the key role played by water molecules in mediating H‐bond formation in β₂‐sheets commonly found in amyloidogenic glutamic acid–rich aggregates.     

The role of proline‐containing peptide triads in β‐sheet formation: A kinetic study

The design of biomimetic materials through molecular self‐assembly is a growing area of modern nanotechnology. With problems of protein folding, self‐assembly, and sequence–structure relationships as essential in nanotechnology as in biology, the effect of the nucleation of β‐hairpin formation by proline on the folding process has been investigated in model studies. Previously such studies were limited to investigations of the influence of proline on the formation of turns in short peptide sequences. The effect of proline‐based triads on the folding of an 11‐kDa amyloidogenic peptide GH₆[(GA)₃GY(GA)₃GE]₈GAH₆ ( YE8 ) was investigated by selective substitution of the proline‐substituted triads at the γ‐turn sites. The folding and fibrillation of the singly proline‐substituted polypeptides, e.g., GH6? [(GA)3GY(GA)3GE]7(GA)3GY(GA)3PD? GAH6 ( 8PD ), and doubly proline‐substituted polypeptides, e.g., GH6? [(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD[(GA)3GY(GA)3GE]3(GA)3GY(GA)3PD? GAH6 ( 4,8PD ), were directly monitored by circular dichroism and deep UV resonance Raman and fluorescence spectroscopies. These findings were used to identify the essential folding domains, i.e., the minimum number of β‐strands necessary for stable folding. These experimental findings may be especially useful in the design and construction of peptidic materials for a wide range of applications as well as in understanding the mechanisms of folding critical to fibril formation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 339–350, 2015.     

Mechanism of pH‐sensitive polymer‐assisted protein refolding and its application in TGF‐β1 and KGF‐2

Refolding of proteins at high concentrations often results in non‐productive aggregation. This study, through a unique combination of spectroscopic and chromatographic analyzes, provides biomolecular evidence to demonstrate the ability of Eudragit S‐100, a pH‐responsive polymer, to enhance refolding of denatured‐reduced lysozyme at high concentrations. The addition of Eudragit in the refolding buffer significantly increases lysozyme refolding yield to 75%, when dilution refolding was conducted at 1 mg/mL lysozyme. This study shows evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit polymer during refolding. This ionic complexing of Eudragit and lysozyme appears to shield exposed hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic‐driven aggregation of the molecules. Importantly, results from this study show that the Eudragit‐lysozyme bioconjugation does not compromise refolded protein structure, and that the polymer can be readily dissociated from the protein by ion exchange chromatography. The strategy was also applied to refolding of TGF‐β1 and KGF‐2. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Interaction of a β‐sheet breaker peptide with lipid membranes

Aggregation of β‐amyloid peptides into senile plaques has been identified as one of the hallmarks of Alzheimer's disease. An attractive therapeutic strategy for Alzheimer's disease is the inhibition of the soluble β‐amyloid aggregation using synthetic β‐sheet breaker peptides that are capable of binding Aβ but are unable to become part of a β‐sheet structure. As the early stages of the Aβ aggregation process are supposed to occur close to the neuronal membrane, it is strategic to define the β‐sheet breaker peptide positioning with respect to lipid bilayers. In this work, we have focused on the interaction between the β‐sheet breaker peptide acetyl‐LPFFD‐amide, iAβ5p, and lipid membranes, studied by ESR spectroscopy, using either peptides alternatively labeled at the C‐ and at the N‐terminus or phospholipids spin‐labeled in different positions of the acyl chain. Our results show that iAβ5p interacts directly with membranes formed by the zwitterionic phospholipid dioleoyl phosphatidylcholine and this interaction is modulated by inclusion of cholesterol in the lipid bilayer formulation, in terms of both peptide partition coefficient and the solubilization site. In particular, cholesterol decreases the peptide partition coefficient between the membrane and the aqueous medium. Moreover, in the absence of cholesterol, iAβ5p is located between the outer part of the hydrophobic core and the external hydrophilic layer of the membrane, while in the presence of cholesterol it penetrates more deeply into the lipid bilayer. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Interaction investigations of crustacean β‐GBP recognition toward pathogenic microbial cell membrane and stimulate upon prophenoloxidase activation

In invertebrates, crustaceans' immune system consists of pattern recognition receptors (PRRs) instead of immunoglobulin's, which involves in the microbial recognition and initiates the protein–ligand interaction between hosts and pathogens. In the present study, PRRs namely β‐1,3 glucan binding protein (β‐GBP) from mangrove crab Episesarma tetragonum and its interactions with the pathogens such as bacterial and fungal outer membrane proteins (OMP) were investigated through microbial aggregation and computational interaction studies. Molecular recognition and microbial aggregation results of Episesarma tetragonum β‐GBP showed the specific binding affinity toward the fungal β‐1,3 glucan molecule when compared to other bacterial ligands. Because of this microbial recognition, prophenoloxidase activity was enhanced and triggers the innate immunity inside the host animal. Our findings disclose the role of β‐GBP in molecular recognition, host–pathogen interaction through microbial aggregation, and docking analysis. In vitro results were concurred with the in silico docking, and molecular dynamics simulation analysis. This study would be helpful to understand the molecular mechanism of β‐GBP and update the current knowledge on the PRRs of crustaceans. Copyright © 2014 John Wiley & Sons, Ltd.     

Conserved buried water molecules enable the β‐trefoil architecture

Available high‐resolution crystal structures for the family of β‐trefoil proteins in the structural databank were queried for buried waters. Such waters were classified as either: (a) unique to a particular domain, family, or superfamily or (b) conserved among all β‐trefoil folds. Three buried waters conserved among all β‐trefoil folds were identified. These waters are related by the threefold rotational pseudosymmetry characteristic of this protein architecture (representing three instances of an identical structural environment within each repeating trefoil‐fold motif). The structural properties of this buried water are remarkable and include: residing in a cavity space no larger than a single water molecule, exhibiting a positional uncertainty (i.e., normalized B‐factor) substantially lower than the average Cα atom, providing essentially ideal H‐bonding geometry with three solvent‐inaccessible main chain groups, simultaneously serving as a bridging H‐bond for three different β‐strands at a point of secondary structure divergence, and orienting conserved hydrophobic side chains to form a nascent core‐packing group. Other published work supports an interpretation that these interactions are key to the formation of an efficient folding nucleus and folded thermostability. The fundamental threefold symmetric structural element of the β‐trefoil fold is therefore, surprisingly, a buried water molecule.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.