Crystallization of a fragment of human fibronectin: Introduction of methionine by site‐directed mutagenesis to allow phasing via selenomethionine

Leahy, Daniel J.; Erickson, Harold P.; Aukhil, Ikramuddin; Joshi, Pallavi; Hendrickson, Wayne A.

doi:10.1002/prot.340190107

Cited by 86 publications

(57 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Peak fractions were pooled and dialyzed overnight at 4°C into the minimal crystallization buffer and concentrated to a final protein concentration of 5 mg͞ml. For selenomethionyl protein production, the Met auxotroph E. coli strain B834 was used and grown as previously described (31). Purification of selenomethionyl LD was performed as described above except for the addition of 5 mM methionine to the purification buffers as a supplemental reducing agent.…”

Section: Methodsmentioning

confidence: 99%

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

Credle

Finer-Moore

Papa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

494

473

View full text Add to dashboard Cite

Unfolded proteins in the endoplasmic reticulum (ER) activate the ER transmembrane sensor Ire1 to trigger the unfolded protein response (UPR), a homeostatic signaling pathway that adjusts ER protein folding capacity according to need. Ire1 is a bifunctional enzyme, containing cytoplasmic kinase and RNase domains whose roles in signal transduction downstream of Ire1 are understood in some detail. By contrast, the question of how its ER-luminal domain (LD) senses unfolded proteins has remained an enigma. The 3.0-Å crystal structure and consequent structure-guided functional analyses of the conserved core region of the LD (cLD) leads us to a proposal for the mechanism of response. cLD exhibits a unique protein fold and is sufficient to control Ire1 activation by unfolded proteins. Dimerization of cLD monomers across a large interface creates a shared central groove formed by ␣-helices that are situated on a ␤-sheet floor. This groove is reminiscent of the peptide binding domains of major histocompatibility complexes (MHCs) in its gross architecture. Conserved amino acid side chains in Ire1 that face into the groove are shown to be important for UPR activation in that their mutation reduces the response. Mutational analyses suggest that further interaction between cLD dimers is required to form higher-order oligomers necessary for UPR activation. We propose that cLD directly binds unfolded proteins, which changes the quaternary association of the monomers in the membrane plane. The changes in the ER lumen in turn position Ire1 kinase domains in the cytoplasm optimally for autophosphorylation to initiate the UPR.Ire1 ͉ unfolded protein response ͉ MHC ͉ protein folding ͉ secretory pathway

show abstract

Section: Methodsmentioning

confidence: 99%

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

Credle

Finer-Moore

Papa

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

494

473

View full text Add to dashboard Cite

show abstract

“…Recombinant FUD (functional upstream domain, clone pUR4) encompassing the non-repeated domain plus 6 amino acids from the first repeat region of F1, a FN-binding adhesin from S. pyogenes was prepared as a recombinant His-tagged protein as described previously . The plasmid for expression of repeats III 7-10 of FN was a generous gift from Harold Erickson (Duke University) and used to produce purified III 7-10 as described previously (Leahy et al, 1994;Zhang et al, 1999).…”

Section: Methodsmentioning

confidence: 99%

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

2006

View full text Add to dashboard Cite

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5min following ligand addition at concentrations as low as 5nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with α 6 and β 1 integrin subunits at the base of the extensions. α 5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectinfibronectin interactions during progression of assembly.

show abstract

“…All mutants and constructs were expressed in this manner, except for selenomethionine-substituted protein. Selenomethionine-substituted protein in which all Met residues were replaced with selenomethionines was expressed as discussed by Hendrickson (Leahy et al, 1994).…”

Section: Expression Of Partial Vp14mentioning

confidence: 99%

Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

et al. 2010

View full text Add to dashboard Cite

The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-Å resolution, provides both insight into the determinants of regio-and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

show abstract

Crystallization of a fragment of human fibronectin: Introduction of methionine by site‐directed mutagenesis to allow phasing via selenomethionine

Cited by 86 publications

References 25 publications

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

On the mechanism of sensing unfolded protein in the endoplasmic reticulum

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

Contact Info

Product

Resources

About