Tight Dimer Research Articles

Crystal structure of blue laccase BP76, a unique termite suicidal defense weapon

Aging workers of the termite Neocapritermes taracua can defend their colony by sacrificing themselves by body rupture, mixing the externally stored blue laccase BP76 with hydroquinones to produce a sticky liquid rich in toxic benzoquinones. Here, we describe the crystal structure of BP76 isolated from N.taracua in its native form. The structure reveals several stabilization strategies, including compact folding, glycosylation, and flexible loops with disulfide bridges and tight dimer interface. The remarkable stability of BP76 maintains its catalytic activity in solid state during the lifespan of N.taracua workers, providing old workers with an efficient defensive weapon to protect their colony.

Posttranslational modifications of cyclic AMP-dependent protein kinase a affect its binding to the CFTR anion channel.

CFTR is the only ion channel member in the ABC protein superfamily. Mutations of the protein cause malfunction of the pore or retention of the protein in the endoplasmic reticulum leading to the systemic disease cystic fibrosis, the most common lethal genetic disorder in the Caucasian population. CFTR activity relies on an irreversible gating mechanism which involves a cycle of ATP binding and hydrolysis at its cytosolic nucleotide binding domains (NBDs). Upon binding of two molecules of ATP the NBDs form a tight NBD dimer which triggers opening of the channel pore, while ATP hydrolysis closes the channel. ATP-dependent opening of the pore strongly depends on phosphorylation of the channel's regulatory (R) domain by cAMP-dependent protein kinase (PKA). At physiological ATP concentrations the NBDs of the channel are saturated by ligand, hence PKA phosphorylation plays the primary regulatory role. Our group has previously shown (PNAS 117:21740) that direct physical binding of PKA activates CFTR channels independently of, and additively to, phosphorylation. We hypothesized that PKA binding prevents its substrate, the R domain, from returning into an inhibitory position wedged in between the two NBDs, thereby shifting the closed-open equilibrium towards open. In contrast to the protein isolated from bovine heart, heterologously expressed PKA lacks such a binding effect. We therefore studied posttranslational modifications of PKA, and investigated their effects on CFTR activation by comparing the recombinant and tissue-isolated PKA isoforms. Results of the ongoing research will be presented at the meeting.

The Annexin A2/S100A10 Complex: The Mutualistic Symbiosis of Two Distinct Proteins.

Mutualistic symbiosis refers to the symbiotic relationship between individuals of different species in which both individuals benefit from the association. S100A10, a member of the S100 family of Ca2+-binding proteins, exists as a tight dimer and binds two annexin A2 molecules. This association forms the annexin A2/S100A10 complex known as AIIt, and modifies the distinct functions of both proteins. Annexin A2 is a Ca2+-binding protein that binds F-actin, phospholipid, RNA, and specific polysaccharides such as heparin. S100A10 does not bind Ca2+, but binds tPA, plasminogen, certain plasma membrane ion channels, neurotransmitter receptors, and the structural scaffold protein, AHNAK. S100A10 relies on annexin A2 for its intracellular survival: in the absence of annexin A2, it is rapidly destroyed by ubiquitin-dependent and independent proteasomal degradation. Annexin A2 requires S100A10 to increase its affinity for Ca2+, facilitating its participation in Ca2+-dependent processes such as membrane binding. S100A10 binds tissue plasminogen activator and plasminogen, and promotes plasminogen activation to plasmin, which is a process stimulated by annexin A2. In contrast, annexin A2 acts as a plasmin reductase and facilitates the autoproteolytic destruction of plasmin. This review examines the relationship between annexin A2 and S100A10, and how their mutualistic symbiosis affects the function of both proteins.

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA

The nucleocapsid phosphoprotein N plays critical roles in multiple processes of the severe acute respiratory syndrome coronavirus 2 infection cycle: it protects and packages viral RNA in N assembly, interacts with the inner domain of spike protein, binds to structural membrane (M) protein during virion packaging and maturation, and to proteases causing replication of infective virus particle. Even with its importance, very limited biophysical studies are available on the N protein because of its high level of disorder, high propensity for aggregation, and high susceptibility for autoproteolysis. Here, we successfully prepare the N protein and a 1000-nucleotide fragment of viral RNA in large quantities and purity suitable for biophysical studies. A combination of biophysical and biochemical techniques demonstrates that the N protein is partially disordered and consists of an independently folded RNA-binding domain and a dimerization domain, flanked by disordered linkers. The protein assembles as a tight dimer with a dimerization constant of sub-micromolar but can also form transient interactions with other N proteins, facilitating larger oligomers. NMR studies on the ∼100-kDa dimeric protein identify a specific domain that binds 1–1000-nt RNA and show that the N-RNA complex remains highly disordered. Analytical ultracentrifugation, isothermal titration calorimetry, multiangle light scattering, and cross-linking experiments identify a heterogeneous mixture of complexes with a core corresponding to at least 70 dimers of N bound to 1–1000 RNA. In contrast, very weak binding is detected with a smaller construct corresponding to the RNA-binding domain using similar experiments. A model that explains the importance of the bivalent structure of N to its binding on multivalent sites of the viral RNA is presented.

Conserved Conformational Changes in the Regulation of Mycobacterium tuberculosis MazEF-mt1

Toxin-antitoxin (TA) systems, which regulate many important cellular processes, are abundantly present in prokaryotic organisms. MazEF is a common type of TA system implicated in the formation of "persisters cells" of the pathogen Mycobacterium tuberculosis, which contains 10 such systems. However, the exact function and inhibition mode of each MazF protein are not quite understood. Here, we report four high-resolution crystal structures of MazF-mt1 in various forms, including one in complex with MazE-mt1. The toxin displayed two unique interlocked loops that allow the formation of a tight dimer. These loops would open upon interacting with the MazE-mt1 antitoxin mediated by the last two helices of MazE-mt1. With our structure-based design, a mutant that could bind to the antitoxin with an enhanced affinity was produced. Combined crystallographic and biochemical studies further revealed that the binding affinity of MazE-mt1 to MazF-mt1 was mainly attributed to its α3 helical region, while the terminal helix η1 contributes very little or even negatively to the association of the pair, in stark contrast to the MazEF-mt9 system. This study provides structural insight into the binding mode and the inhibition mechanism of the MazE/F-mt1 TA pair, which may reflect the functional differences between different TA systems.

U2AF1 Driver Mutations in Hematopoietic Disorders Alter but Do Not Abrogate RNA Binding and Enlighten Structural Dependencies of the U2AF-RNA Complex

Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.

Molecular architecture of the Bardet–Biedl syndrome protein 2-7-9 subcomplex

Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its α-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation.

Structural basis of small RNA hydrolysis by oligoribonuclease (CpsORN) from Colwellia psychrerythraea strain 34H

Cells regulate their intracellular mRNA levels by using specific ribonucleases. Oligoribonuclease (ORN) is a 3′–5′ exoribonuclease for small RNA molecules, important in RNA degradation and re-utilisation. However, there is no structural information on the ligand-binding form of ORNs. In this study, the crystal structures of oligoribonuclease from Colwellia psychrerythraea strain 34H (CpsORN) were determined in four different forms: unliganded-structure, thymidine 5′-monophosphate p-nitrophenyl ester (pNP-TMP)-bound, two separated uridine-bound, and two linked uridine (U-U)-bound forms. The crystal structures show that CpsORN is a tight dimer, with two separated active sites and one divalent metal cation ion in each active site. These structures represent several snapshots of the enzymatic reaction process, which allowed us to suggest a possible one-metal-dependent reaction mechanism for CpsORN. Moreover, the biochemical data support our suggested mechanism and identified the key residues responsible for enzymatic catalysis of CpsORN.

The structure of iPLA2\u03b2 reveals dimeric active sites and suggests mechanisms of regulation and localization

Calcium-independent phospholipase A2β (iPLA2β) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson’s disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2β-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2β that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA2β cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA2β in the brain.

Structural and biophysical characterization of Rv3716c, a hypothetical protein from Mycobacterium tuberculosis

Latent tuberculosis (TB) is the main hurdle in reaching the goal of “Stop TB 2050”. Tuberculin skin and Interferon-gamma release assay tests used currently for the diagnosis of TB infection cannot distinguish between active disease and latent tuberculosis infection (LTBI) and hence new and sensitive protein markers need to be identified for the diagnosis. A protein Rv3716c from Mycobacterium tuberculosis (MtbRv3716c) has been identified as a potential surrogate marker for the diagnosis of LTBI. Here, we present characterization of MtbRv3716c (∼13 kDa) using both biophysical and X-Ray crystallographic methods. EMSA study showed that MtbRv3716c binds to double stranded DNA. X-ray diffraction data collected on a crystal of MtbRv3716c at 1.9 Å resolution was used for structure determination using the molecular replacement method. Significant electron density was not observed for the N-terminal 21 and C-terminal 41 residues in the final electron density map. The C- terminal disordered region is proline rich and displays characteristics of intrinsically disordered proteins. Although the crystal asymmetric unit contained a protomer, a tight dimer could be generated by the application of the crystal two-fold symmetry parallel to the b axis. Packing of dimers in the crystal is mediated by a cadmium ion (Cd2+) occurring at the interface of two dimers. Molecular packing analysis reveals large cavities that are probably occupied by the disordered segments of the N- and C-termini. Structural comparison with other homologous hypothetical DNA binding proteins (PDB codes: 1PUG, 1YBX) highlights structural features that might be significant for DNA binding.

Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a Gram-negative bacterium and causative agent of Crown Gall disease that infects a variety of economically important plants. The annotated A. tumefaciens genome contains 10 putative dapA genes, which code for dihydrodipicolinate synthase (DHDPS). However, we have recently demonstrated that only one of these genes (dapA7) encodes a functional DHDPS. The function of the other nine putative dapA genes is yet to be determined. Here, we demonstrate using bioinformatics that the product of the dapA5 gene (DapA5) possesses all the catalytic residues canonical to 2-keto-3-deoxygluconate (KDG) aldolase, which is a class I aldolase involved in glucose metabolism. We therefore expressed, purified, and characterized recombinant DapA5 using mass spectrometry, circular dichroism spectroscopy, analytical ultracentrifugation, and enzyme kinetics. The results show that DapA5 (1) adopts an α/β structure consistent with the TIM-barrel fold of KDG aldolases, (2) possesses KDG aldolase enzyme activity, and (3) exists as a tight dimer in solution. This study shows for the first time that dapA5 from A. tumefaciens encodes a functional dimeric KDG aldolase.

Computational design of a symmetrical \u03b2-trefoil lectin with cancer cell binding activity

Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial β-trefoil lectin that recognises Burkitt’s lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called “Mitsuba”, is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning “three-leaf” in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galβ(1,4)Glc) on the surface, but the cytotoxicity is abolished.

Potent Inhibition of 3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) Synthase by DAHP Oxime, a Phosphate Group Mimic.

3-Deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyzes the first step in the shikimate pathway. It catalyzes an aldol-like reaction of phosphoenolpyruvate (PEP) with erythrose 4-phosphate (E4P) to form DAHP. The kinetic mechanism was rapid equilibrium sequential ordered ter ter, with the essential divalent metal ion, Mn2+, binding first, followed by PEP and E4P. DAHP oxime, in which an oxime group replaces the keto oxygen, was a potent inhibitor, with Ki = 1.5 ± 0.4 μM, though with residual activity at high inhibitor concentrations. It displayed slow-binding inhibition with a residence time, tR, of 83 min. The crystal structure revealed that the oxime functional group, combined with two crystallographic waters, bound at the same location in the catalytic center as the phosphate group of the tetrahedral intermediate. DAHP synthase has a dimer-of-dimers homotetrameric structure, and DAHP oxime bound to only one subunit of each tight dimer. Inhibitor binding was competitive with respect to all three substrates in the subunits to which it bound. DAHP oxime did not overlap with the metal binding site, so the cause of their mutually exclusive binding was not clear. Similarly, there was no obvious structural reason for inhibitor binding in only two subunits; however, changes in global hydrogen/deuterium exchange showed large scale changes in protein dynamics upon inhibitor binding. The kcat value for the residual activity at high inhibitor concentrations was 3-fold lower, and the apparent KM,E4P value decreased at least 10-fold. This positive cooperativity of binding between DAHP oxime in subunits B and C, and E4P in subunits A and D appears to be the dominant cause for incomplete inhibition at high inhibitor concentrations. In spite of its lack of obvious structural similarity to phosphate, the oxime and crystallographic waters acted as a small, neutral phosphate mimic.

Crystal structure of MytiLec, a galactose-binding lectin from the mussel Mytilus galloprovincialis with cytotoxicity against certain cancer cell types.

MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Galα(1,4)Galβ(1,4)Glcα1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Galα(1,6)Glc, and the trisaccharide globotriose, Galα(1,4)Galβ(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt’s lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the β-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding, and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects.

Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors.

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2’,4,4’-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0181-1) contains supplementary material, which is available to authorized users.

Structure and Biophysical Characterization of the S-Adenosylmethionine-dependent O-Methyltransferase PaMTH1, a Putative Enzyme Accumulating during Senescence of Podospora anserina

Low levels of reactive oxygen species (ROS) act as important signaling molecules, but in excess they can damage biomolecules. ROS regulation is therefore of key importance. Several polyphenols in general and flavonoids in particular have the potential to generate hydroxyl radicals, the most hazardous among all ROS. However, the generation of a hydroxyl radical and subsequent ROS formation can be prevented by methylation of the hydroxyl group of the flavonoids. O-Methylation is performed by O-methyltransferases, members of the S-adenosyl-l-methionine (SAM)-dependent O-methyltransferase superfamily involved in the secondary metabolism of many species across all kingdoms. In the filamentous fungus Podospora anserina, a well established aging model, the O-methyltransferase (PaMTH1) was reported to accumulate in total and mitochondrial protein extracts during aging. In vitro functional studies revealed flavonoids and in particular myricetin as its potential substrate. The molecular architecture of PaMTH1 and the mechanism of the methyl transfer reaction remain unknown. Here, we report the crystal structures of PaMTH1 apoenzyme, PaMTH1-SAM (co-factor), and PaMTH1-S-adenosyl homocysteine (by-product) co-complexes refined to 2.0, 1.9, and 1.9 Å, respectively. PaMTH1 forms a tight dimer through swapping of the N termini. Each monomer adopts the Rossmann fold typical for many SAM-binding methyltransferases. Structural comparisons between different O-methyltransferases reveal a strikingly similar co-factor binding pocket but differences in the substrate binding pocket, indicating specific molecular determinants required for substrate selection. Furthermore, using NMR, mass spectrometry, and site-directed active site mutagenesis, we show that PaMTH1 catalyzes the transfer of the methyl group from SAM to one hydroxyl group of the myricetin in a cation-dependent manner.

Lipid-directed vinculin dimerization.

Vinculin localizes to cellular adhesions where it regulates motility, migration, development, wound healing, and response to force. Importantly, vinculin loss results in cancer phenotypes, cardiovascular disease, and embryonic lethality. At the plasma cell membrane, the most abundant phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), binds the vinculin tail domain, Vt, and triggers homotypic and heterotypic interactions that amplify binding of vinculin to the actin network. Binding of PIP2 to Vt is necessary for maintaining optimal focal adhesions, for organizing stress fibers, for cell migration and spreading, and for the control of vinculin dynamics and turnover of focal adhesions. While the recently determined Vt/PIP2 crystal structure revealed the conformational changes occurring upon lipid binding and oligomerization, characterization of PIP2-induced vinculin oligomerization has been challenging in the adhesion biology field. Here, via a series of novel biochemical assays not performed in previous studies that relied on chemical cross-linking, we characterize the PIP2-induced vinculin oligomerization. Our results show that Vt/PIP2 forms a tight dimer with Vt or with the muscle-specific vinculin isoform, metavinculin, at sites of adhesion at the cell membrane. Insight into how PIP2 regulates clustering and into mechanisms that regulate cell adhesion allows the development for a more definite sensor for PIP2, and our developed techniques can be applied generally and thus open the door for the characterization of many other protein/PIP2 complexes under physiological conditions.

Structural and Functional Study of d-Glucuronyl C5-epimerase

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Modeling of particle interactions in magnetorheological elastomers

The interaction between two particles made of an isotropic linearly polarizable magnetic material and embedded in an elastomer matrix is studied. In this case, when an external field is imposed, the magnetic attraction of the particles, contrary to point dipoles, is almost wraparound. The exact solution of the magnetic problem in the linear polarization case, although existing, is not practical; to circumvent its use, an interpolation formula is proposed. One more interpolation expression is developed for the resistance of the elastic matrix to the field-induced particle displacements. Minimization of the total energy of the pair reveals its configurational bistability in a certain field range. One of the possible equilibrium states corresponds to the particles dwelling at a distance, the other—to their collapse in a tight dimer. This mesoscopic bistability causes magnetomechanical hysteresis which has important implications for the macroscopic behavior of magnetorheological elastomers.