Zinc Ions Induce the Unfolding and Self-Association of Boar Spermadhesin PSP-I, a Protein with a Single CUB Domain Architecture, and Promote Its Binding to Heparin†

Abstract The large, secreted glycoprotein reelin regulates embryonic brain development as well as adult brain functions. Although reelin binds to its receptors via its central part, the N-terminal region directs multimer formation and is critical for efficient signal transduction. In fact, the inhibitory antibody CR-50 interacts with the N-terminal region and prevents higher-order multimerization and signaling. Reelin is a multidomain protein in which the central part is composed of eight characteristic repeats, named reelin repeats, each of which is further divided by insertion of an EGF module into two subrepeats. In contrast, the N-terminal region shows unique “irregular” domain architecture since it comprises three consecutive subrepeats without the intervening EGF module. Here we determined the crystal structure of the murine reelin fragment named RX-R1 including the irregular region and the first reelin repeat at 2.0 Å resolution. The overall structure of RX-R1 has a branched Y-shaped form. Interestingly, two incomplete subrepeats cooperatively form one entire subrepeat structure, though an additional subrepeat is inserted between them. We further reveal that Arg335 of RX-R1 is crucial for binding CR-50. A possible self-association mechanism via the N-terminal region is proposed based on our results.

Download Full-text

Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association

Biochemical Journal ◽

10.1042/bj20040253 ◽

2004 ◽

Vol 381 (2) ◽

pp. 547-559 ◽

Cited By ~ 16

Author(s):

Soren PRAG ◽

Georgina D. M. COLLETT ◽

Josephine C. ADAMS

Keyword(s):

Cellular Localization ◽

Domain Architecture ◽

Wild Type ◽

Intracellular Protein ◽

Repeat Proteins ◽

Repeat Domain ◽

Functional Involvement ◽

Self Association ◽

Cis And Trans ◽

Structural Parallel

Muskelin is an intracellular protein with a C-terminal kelch-repeat domain that was initially characterized as having functional involvement in cell spreading on the extracellular matrix glycoprotein thrombospondin-1. As one approach to understanding the functional properties of muskelin, we have combined bioinformatic and biochemical studies. Through analysis of a new dataset of eight animal muskelins, we showed that the N-terminal region of the polypeptide corresponds to a predicted discoidin-like domain. This domain architecture is conserved in fungal muskelins and reveals a structural parallel between the muskelins and certain extracellular fungal galactose oxidases, although the phylogeny of the two groups appears distinct. In view of the fact that a number of kelch-repeat proteins have been shown to self-associate, co-immunoprecipitation, protein pull-down assays and studies of cellular localization were carried out with wild-type, deletion mutant and point mutant muskelins to investigate the roles of the discoidin-like and kelch-repeat domains. We obtained evidence for cis- and trans-interactions between the two domains. These studies provide evidence that muskelin self-associates through a head-to-tail mechanism involving the discoidin-like domain.

Download Full-text

Tubulin structure at intermediate resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140634 ◽

1995 ◽

Vol 53 ◽

pp. 852-853

Author(s):

K. H. Downing ◽

S. G. Wolf ◽

E. Nogales

Keyword(s):

High Resolution ◽

Structural Data ◽

Therapeutic Drug ◽

Zinc Ions ◽

Two Dimensional ◽

X Ray Diffraction ◽

X Ray ◽

Negative Stain ◽

Functional Mechanisms ◽

Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

Download Full-text

Aggregated, Conformationally Changed Fibrinogen Exposes the Stimulating Sites for t-PA-Catalysed Plasminogen Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650269 ◽

1996 ◽

Vol 75 (02) ◽

pp. 326-331 ◽

Cited By ~ 7

Author(s):

Unni Haddeland ◽

Knut Sletten ◽

Anne Bennick ◽

Willem Nieuwenhuizen ◽

Frank Brosstad

Keyword(s):

Conformational Changes ◽

Gel Permeation Chromatography ◽

Tissue Type ◽

Plasminogen Activation ◽

Chromogenic Substrate ◽

Type Plasminogen Activator ◽

Gel Permeation ◽

Self Association ◽

Fibrin Monomers ◽

Stimulation Of

SummaryThe present paper shows that conformationally changed fibrinogen can expose the sites Aα-(148-160) and γ-(312-324) involved in stimulation of the tissue-type plasminogen activator (t-PA)-catalysed plasminogen activation. The exposure of the stimulating sites was determined by ELISA using mABs directed to these sites, and was shown to coincide with stimulation of t-PA-catalysed plasminogen activation as assessed in an assay using a chromogenic substrate for plasmin. Gel permeation chromatography of fibrinogen conformationally changed by heat (46.5° C for 25 min) demonstrated the presence of both aggregated and monomeric fibrinogen. The aggregated fibrinogen, but not the monomeric fibrinogen, had exposed the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation. Fibrinogen subjected to heat in the presence of 3 mM of the tetrapeptide GPRP neither aggregates nor exposes the rate-enhancing sites. Thus, aggregation and exposure of t-PA-stimulating sites in fibrinogen seem to be related phenomena, and it is tempting to believe that the exposure of stimulating sites is a consequence of the conformational changes that occur during aggregation, or self-association. Fibrin monomers kept in a monomeric state by a final GPRP concentration of 3 mM do not expose the epitopes Aα-(148-160) and γ-(312-324) involved in t-PA-stimulation, whereas dilution of GPRP to a concentration that is no longer anti-polymerizing, results in exposure of these sites. Consequently, the exposure of t-PA-stimulating sites in fibrin as well is due to the conformational changes that occur during selfassociation.

Download Full-text