Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation.

Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro, which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5' labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.

Dysregulation of Hepatitis B Virus Nucleocapsid Assembly in vitro by RNA-binding Small Ligands.

RNA sequences/motifs dispersed across the genome of Hepatitis B Virus regulate formation of nucleocapsid-like particles (NCPs) by core protein (Cp) in vitro, in an epsilon/polymerase-independent fashion. These multiple RNA Packaging Signals (PSs) can each form stem-loops encompassing a Cp-recognition motif, -RGAG-, in their loops. Drug-like molecules that bind the most important of these PS sites for NCP assembly regulation with nanomolar affinities, were identified by screening an immobilized ligand library with a fluorescently-labelled, RNA oligonucleotide encompassing this sequence. Sixty-six of these "hits", with affinities ranging from low nanomolar to high micromolar, were purchased as non-immobilized versions. Their affinities for PSs and effects on NCP assembly were determined in vitro by Surface Plasmon Resonance. High-affinity ligand binding is dependent on the presence of an -RGAG- motif within the loop of the PS, consistent with ligand cross-binding between PS sites. Simple structure-activity relationships show that it is also dependent on the presence of specific functional groups in these ligands. Some compounds are potent inhibitors of in vitro NCP assembly at nanomolar concentrations. Despite appropriate logP values, these ligands do not inhibit HBV replication in cell culture. However, modelling confirms the potential of using PS-binding ligands to target NCP assembly as a novel anti-viral strategy. This also allows for computational exploration of potential synergic effects between anti-viral ligands directed at distinct molecular targets in vivo. HBV PS-regulated assembly can be dysregulated by novel small molecule RNA-binding ligands opening a novel target for developing directly-acting anti-virals against this major pathogen.

Tuning the rate of aggregation of hIAPP into amyloid using small-molecule modulators of assembly.

Human islet amyloid polypeptide (hIAPP) self-assembles into amyloid fibrils which deposit in pancreatic islets of type 2 diabetes (T2D) patients. Here, we applied chemical kinetics to study the mechanism of amyloid assembly of wild-type hIAPP and its more amyloidogenic natural variant S20G. We show that the aggregation of both peptides involves primary nucleation, secondary nucleation and elongation. We also report the discovery of two structurally distinct small-molecule modulators of hIAPP assembly, one delaying the aggregation of wt hIAPP, but not S20G; while the other enhances the rate of aggregation of both variants at substoichiometric concentrations. Investigation into the inhibition mechanism(s) using chemical kinetics, native mass spectrometry, fluorescence titration, SPR and NMR revealed that the inhibitor retards primary nucleation, secondary nucleation and elongation, by binding peptide monomers. By contrast, the accelerator predominantly interacts with species formed in the lag phase. These compounds represent useful chemical tools to study hIAPP aggregation and may serve as promising starting-points for the development of therapeutics for T2D.

A Comparative Assessment Study of Known Small-molecule GPVI Modulators.

The GPVI platelet receptor was recently validated as a safe antiplatelet target for the treatment of thrombosis using several peptidic modulators. In contrast, few weakly potent small-molecule GPVI antagonists have been reported. Those that have been published often lack evidence for target engagement, and their biological efficacy cannot be compared because of the natural donor variability associated with the assays implemented. Herein, we present the first side-by-side assessment of the reported GPVI small-molecule modulators. We have characterized their functional activities on platelet activation and aggregation using flow cytometry as well as light transmission and electrical impedance aggregometry. We also utilized microscale thermophoresis (MST) and saturation transfer difference (STD) NMR to validate GPVI binding and have used this along with molecular modeling to suggest potential binding interactions. We conclude that of the compounds examined, losartan and compound 5 are currently the most viable GPVI modulators.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region.

OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.

Mutations in Spliceosomal Genes PPIL1 and PRP17 Cause Neurodegenerative Pontocerebellar Hypoplasia with Microcephaly.

Autosomal-recessive cerebellar hypoplasia and ataxia constitute a group of heterogeneous brain disorders caused by disruption of several fundamental cellular processes. Here, we identified 10 families showing a neurodegenerative condition involving pontocerebellar hypoplasia with microcephaly (PCHM). Patients harbored biallelic mutations in genes encoding the spliceosome components Peptidyl-Prolyl Isomerase Like-1 (PPIL1) or Pre-RNA Processing-17 (PRP17). Mouse knockouts of either gene were lethal in early embryogenesis, whereas PPIL1 patient mutation knockin mice showed neuron-specific apoptosis. Loss of either protein affected splicing integrity, predominantly affecting short and high GC-content introns and genes involved in brain disorders. PPIL1 and PRP17 form an active isomerase-substrate interaction, but we found that isomerase activity is not critical for function. Thus, we establish disrupted splicing integrity and "major spliceosome-opathies" as a new mechanism underlying PCHM and neurodegeneration and uncover a non-enzymatic function of a spliceosomal proline isomerase.

A missense variant in specificity protein 6 (SP6) is associated with amelogenesis imperfecta.

Amelogenesis is the process of enamel formation. For amelogenesis to proceed, the cells of the inner enamel epithelium (IEE) must first proliferate and then differentiate into the enamel-producing ameloblasts. Amelogenesis imperfecta (AI) is a heterogeneous group of genetic conditions that result in defective or absent tooth enamel. We identified a 2 bp variant c.817_818GC>AA in SP6, the gene encoding the SP6 transcription factor, in a Caucasian family with autosomal dominant hypoplastic AI. The resulting missense protein change, p.(Ala273Lys), is predicted to alter a DNA-binding residue in the first of three zinc fingers. SP6 has been shown to be crucial to both proliferation of the IEE and to its differentiation into ameloblasts. SP6 has also been implicated as an AI candidate gene through its study in rodent models. We investigated the effect of the missense variant in SP6 (p.(Ala273Lys)) using surface plasmon resonance protein-DNA binding studies. We identified a potential SP6 binding motif in the AMBN proximal promoter sequence and showed that wild-type (WT) SP6 binds more strongly to it than the mutant protein. We hypothesize that SP6 variants may be a very rare cause of AI due to the critical roles of SP6 in development and that the relatively mild effect of the missense variant identified in this study is sufficient to affect amelogenesis causing AI, but not so severe as to be incompatible with life. We suggest that current AI cohorts, both with autosomal recessive and dominant disease, be screened for SP6 variants.

Defining the remarkable structural malleability of a bacterial surface protein Rib domain implicated in infection.

Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the ß-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin ß-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

AtSPX1 affects the AtPHR1-DNA-binding equilibrium by binding monomeric AtPHR1 in solution.

Phosphorus is an essential macronutrient for plant growth and is deficient in ∼50% of agricultural soils. The transcription factor phosphate starvation response 1 (PHR1) plays a central role in regulating the expression of a subset of phosphate starvation-induced (PSI) genes through binding to a cis-acting DNA element termed P1BS (PHR1-binding sequences). In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX (Syg1, Pho81, Xpr1) proteins through protein-protein interaction. Here, we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using surface plasmon resonance, a tandem P1BS sequence showed ∼50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA-binding domain and coiled-coil domain of AtPHR1 fused to maltose-binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2× P1BS-binding site, suggesting an important role for protein co-operativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA-binding signal from MBPAtdPHR1. These data suggest that, in the Pi-restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA-binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA-binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression.

The effects of irradiation on the biological and biomechanical properties of an acellular porcine superflexor tendon graft for cruciate ligament repair.

Acellular xenogeneic tissues have the potential to provide 'off-the-shelf' grafts for anterior cruciate ligament (ACL) repair. To ensure that such grafts are sterile following packaging, it is desirable to use terminal sterilization methods. Here, the effects of gamma and electron beam irradiation on the biological and biomechanical properties of a previously developed acellular porcine superflexor tendon (pSFT) were investigated. Irradiation following treatment with peracetic acid was compared to peracetic acid treatment alone and the stability of grafts following long-term storage assessed. Irradiation did not affect total collagen content or biocompatibility (determined using a contact cytotoxicity assay) of the grafts, but slightly increased the amount of denatured collagen in and decreased the thermal denaturation temperature of the tissue in a dose dependant fashion. Biomechanical properties of the grafts were altered by irradiation (reduced ultimate tensile strength and Young's modulus, increased failure strain), but remained superior to reported properties of the native human ACL. Long term storage at 4°C had no negative effects on the grafts. Of all the conditions tested, a dose of minimum 25 kGy of gamma irradiation had least effect on the grafts, suggesting that this dose produces a biocompatible pSFT graft with adequate mechanical properties for ACL repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2477-2486, 2017.

Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4.

AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 µmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 µmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1ß, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis. RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor.

Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2.

Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)-RNA and maturation protein (MP)-RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA.

Trivalent Gd-DOTA reagents for modification of proteins.

The development of novel protein-targeted MRI contrast agents crucially depends on the ability to derivatise suitable targeting moieties with a high payload of relaxation enhancer (e.g., gadolinium(iii) complexes such as Gd-DOTA), without losing affinity for the target proteins. Here, we report robust synthetic procedures for the preparation of trivalent Gd-DOTA reagents with various chemical handles for site-specific modification of biomolecules. The reagents were shown to successfully label proteins through isothiocyanate ligation or through site-specific thiol-maleimide ligation and strain-promoted azide-alkyne cycloaddition.

Epitope detection chromatography: a method to dissect the structural heterogeneity and inter-connections of plant cell-wall matrix glycans.

Plant cell walls are complex, multi-macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell-wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell-wall matrix glycan sub-populations and inter-connections. The method combines chromatographic separations with use of glycan-directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell-wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub-populations and their modulation in A. thaliana organs.

Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors.

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.

Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls.

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG-I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG-I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG-I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.

Staphylococcal biofilm-forming protein has a contiguous rod-like structure.

Staphylococcus aureus and Staphylococcus epidermidis form communities (called biofilms) on inserted medical devices, leading to infections that affect many millions of patients worldwide and cause substantial morbidity and mortality. As biofilms are resistant to antibiotics, device removal is often required to resolve the infection. Thus, there is a need for new therapeutic strategies and molecular data that might assist their development. Surface proteins S. aureus surface protein G (SasG) and accumulation-associated protein (S. epidermidis) promote biofilm formation through their "B" regions. B regions contain tandemly arrayed G5 domains interspersed with approximately 50 residue sequences (herein called E) and have been proposed to mediate intercellular accumulation through Zn(2+)-mediated homodimerization. Although E regions are predicted to be unstructured, SasG and accumulation-associated protein form extended fibrils on the bacterial surface. Here we report structures of E-G5 and G5-E-G5 from SasG and biophysical characteristics of single and multidomain fragments. E sequences fold cooperatively and form interlocking interfaces with G5 domains in a head-to-tail fashion, resulting in a contiguous, elongated, monomeric structure. E and G5 domains lack a compact hydrophobic core, and yet G5 domain and multidomain constructs have thermodynamic stabilities only slightly lower than globular proteins of similar size. Zn(2+) does not cause SasG domains to form dimers. The work reveals a paradigm for formation of fibrils on the 100-nm scale and suggests that biofilm accumulation occurs through a mechanism distinct from the "zinc zipper." Finally, formation of two domains by each repeat (as in SasG) might reduce misfolding in proteins when the tandem arrangement of highly similar sequences is advantageous.