Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex

Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cellulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1Cel6A) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1Cel6A fusion protein had been previously applied. This showed that the SPA-CBM1Cel6A fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.     

Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism

High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen (Populus tremula x Populus tremuloides). Production of the loop deletion variant Tm-NXG1-DeltaYNIIG yielded an enzyme that was structurally similar to Ptt-XET16-34 and had a greatly increased transglycosylation:hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.     

Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.     

Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Peptide-oligonucleotide conjugates form stable and selective complexes with antibody and DNA

The present work demonstrates that the relatively low molecular weight synthetic peptide-oligonucleotide conjugates are capable of stable and selective three-component complex formation with complementary 72-100mer DNA oligonucleotides and a cardiac troponin I monoclonal antibody. Neither the Watson-Crick-type interaction between peptide-oligonucleotide conjugate and DNA nor the conjugate-antibody interaction dramatically hampers the other. These interactions remain selective and specific in the presence of several other conjugates not specific to cardiac troponin I monoclonal antibody as well as in the presence of control 100mer DNA oligonucleotides. The data herein demonstrate the feasibility of the synthetic peptide-oligonucleotide conjugates as convenient molecular tools, e.g., for antibody epitope mapping.     

Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-beta-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.     

A novel specific role for I kappa B kinase complex-associated protein in cytosolic stress signaling

We demonstrate here a novel role for the I kappa B kinase complex-associated protein (IKAP) in the regulation of activation of the mammalian stress response via the c-Jun N-terminal kinase (JNK)-signaling pathway. We cloned IKAP as a JNK-associating protein using the Ras recruitment yeast two-hybrid system. IKAP efficiently and specifically enhanced JNK activation induced by ectopic expression of MEKK1 and ASK1, upstream activators of JNK. Importantly, IKAP also enhanced JNK activation induced by ultraviolet light irradiation as well as treatments with tumor necrosis factor or epidermal growth factor. The JNK association site in IKAP was mapped to the C-terminal part of IKAP. Interestingly, this region is deleted from IKAP expressed in the autonomous nervous system of the patients affected by familial dysautonomia. Ectopic expression of this C-terminal fragment of IKAP was sufficient to support JNK activation. Taken together, our data demonstrate a novel role for IKAP in the regulation of the JNK-mediated stress signaling. Additionally, our results point to a role of JNK signaling in familial dysautonomia and, thus, further support the involvement of JNK signaling in the development, survival, and degeneration of the sensory and autonomic nervous system.     

Parvovirus Induced Alterations in Nuclear Architecture and Dynamics

The nucleus of interphase eukaryotic cell is a highly compartmentalized structure containing the three-dimensional network of chromatin and numerous proteinaceous subcompartments. DNA viruses induce profound changes in the intranuclear structures of their host cells. We are applying a combination of confocal imaging including photobleaching microscopy and computational methods to analyze the modifications of nuclear architecture and dynamics in parvovirus infected cells. Upon canine parvovirus infection, expansion of the viral replication compartment is accompanied by chromatin marginalization to the vicinity of the nuclear membrane. Dextran microinjection and fluorescence recovery after photobleaching (FRAP) studies revealed the homogeneity of this compartment. Markedly, in spite of increase in viral DNA content of the nucleus, a significant increase in the protein mobility was observed in infected compared to non-infected cells. Moreover, analyzis of the dynamics of photoactivable capsid protein demonstrated rapid intranuclear dynamics of viral capsids. Finally, quantitative FRAP and cellular modelling were used to determine the duration of viral genome replication. Altogether, our findings indicate that parvoviruses modify the nuclear structure and dynamics extensively. Intranuclear crowding of viral components leads to enlargement of the interchromosomal domain and to chromatin marginalization via depletion attraction. In conclusion, parvoviruses provide a useful model system for understanding the mechanisms of virus-induced intranuclear modifications.     

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A

We have analyzed the structure and function of the integrin α₁I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α₁I C139S/E317A was a higher avidity collagen binder than α₁I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α₁I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α₂I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α₂I structure, has not changed its position in the activated α₁I variant. During the integrin activation, Glu³³⁵ on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β₁ subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu³³⁵. This indicates that the stabilization of helix 7 into its downward position is not required if the α₁ MIDAS is already open. To conclude, the activated α₁I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg²⁸⁷-Glu³¹⁷ ion pair has just broken during the integrin activation.