Crystallization and preliminary X-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.     

The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins.     

The interrelationship between ligand binding and thermal unfolding of the folate binding protein. The role of self-association and pH

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH 7.4 (pI = 7–8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3 μM holo-FBP showed Tm (76 °C) and molar enthalpy (146 kcal M^− 1) values increasing to 78 °C and 163 kcal M^− 1 at 10 μM holo-FBP, while those of apo-FBP were 55 °C and 105 kcal M^− 1. Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10 μM) in DLS after a step-wise rise in temperature to 78 °C ≈ Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH 5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH 5 (Tm 38 °C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15 M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.     

The complete primary structure of the boar spermadhesin AQN-1, a carbohydrate-binding protein involved in fertilization.

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate-binding proteins on the sperm surface play a pivotal role as counter-receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm-associated zona-pellucida-binding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN-1, a 12-kDa boar-sperm-associated carbohydrate-binding and zona-pellucida-binding protein. The molecular mass of AQN-1 was determined by time-of-flight plasma-desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN-1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN-1 shares extensive structural, as well as functional, similarity with two other boar sperm zona-pellucida-binding proteins, AQN-3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species-specific sperm-egg recognition mechanism.     

Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.     

Influence of the N-terminal domain and divalent cations on self-association and DNA binding by the Saccharomyces cerevisiae TATA binding protein

The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K+, and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the full-length protein.     

Cell cycle localization, dimerization, and binding domain architecture of the telomere protein cPot1

Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.     

Zinc ions promote the binding of factor XII/factor XIIA to acidic phospholipids but have no effect on the binding of high-Mr kininogen

Binding of high-Mr kininogen and factor XII/factor XIIa to phospholipids coated on to polystyrene microtiter plates was investigated by ELISA. Both high-Mr kininogen and factor XII/factor XIIa bound specifically to the phospholipid surface. Binding was observed to negatively charged phospholipids only. The binding of high-Mr kininogen was not affected by the presence of zinc ions. At a surface concentration of 20% phosphatidylinositol phosphate in phosphatidylcholine a dissociation constant (kD) of 10 nM for the binding of high-Mr kininogen was calculated. The amount of bound purified alpha-factor XIIa could be increased 4-5-fold in the presence of zinc ions. The lowest zinc ion concentration giving maximal binding was 0.1 mM. The binding of alpha-factor XIIa was inhibited by high-Mr kininogen. Independent of the presence of zinc ions or high-Mr kininogen, a kD of 7.9 nM was calculated for alpha-factor XIIa binding. The binding of prekallikrein was dependent upon the presence and the concentration of high-Mr kininogen. In plasma containing aprotinin, the binding of high-Mr kininogen was apparently inhibited in the presence of zinc ions, which was a prerequisite for the binding of factor XII. This apparently inhibitory effect of zinc ions on the binding of high-Mr kininogen was probably due to the increased binding of factor XII, which displaced high-Mr kininogen.     

Marked stereoselectivity in the binding of copper ions by heparin. Contrasts with the binding of gadolinium and calcium ions

Heparin forms a complex with cupric ion (Cu2+) at a level of less than or equal to 10(-3) mol of the metal ion per dimeric unit of the polymer, as evidenced by paramagnetic relaxation effects on its 1H- and 13C-n.m.r. spectra. No interaction occurred with heparin derivatives modified either by desulfation of the residues of alpha-L-iduronic acid 2-sulfate, or by hydrolysis of the sulfamino group of the residues of 2-deoxy-2-sulfamino-alpha-D-glucose 6-sulfate, although binding was induced by N-acetylation of the latter derivative. Under the same experimental conditions, no alternative type of glycosyluronic acid structure tested, including the other glycosaminoglycans, showed significant relaxation enhancement by Cu2+. These results are in contrast to those obtained with gadolinium ion (Gd3+), another paramagnetic probe, or with calcium ion (Ca2+), which promotes chemical-shift displacements. The binding selectivities of those two cations are much broader than that of Cu2%, although they also differ notably in their relationship to the structure of heparin.     

Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.

Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation

Protein engineering Phi-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI I27. TNfn3 and TI I27 are members of different protein superfamilies and have no sequence identity but they have the same beta-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI I27. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI I27, TNfn3 unfolds via a force-stabilised intermediate. The key event in forced unfolding in TI I27 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical Phi-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI I27, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force.     

Zinc finger of replication protein A, a non-DNA binding element, regulates its DNA binding activity through redox.

Eukaryotic replication protein A (RPA) is a single-stranded DNA-binding protein with multiple functions in DNA replication, repair, and genetic recombination. RPA contains an evolutionarily conserved 4-cysteine-type zinc finger motif (X(3)CX(2-4)CX(12-15)CX(2)C) that has a potential role in regulation of DNA replication and repair (Dong, J., Park, J-S., and Lee, S-H. (1999) Biochem. J. 337, 311-317 and Lin, Y.-L., Shivji, M. K. K., Chen, C., Kolodner, R., Wood, R. D., and Dutta, A. (1998) J. Biol. Chem. 273, 1453-1461), even though the zinc finger itself is not essential for its DNA binding activity (Kim, D. K., Stigger, E., and Lee, S.-H. (1996) J. Biol. Chem. 271, 15124-15129). Here, we show that RPA single-stranded DNA (ssDNA) binding activity is regulated by reduction-oxidation (redox) through its zinc finger domain. RPA-ssDNA interaction was stimulated 10-fold by the reducing agent, dithiothreitol (DTT), whereas treatment of RPA with oxidizing agent, diazene dicarboxylic acid bis[N,N-dimethylamide] (diamide), significantly reduced this interaction. The effect of diamide was reversed by the addition of excess DTT, suggesting that RPA ssDNA binding activity is regulated by redox. Redox regulation of RPA-ssDNA interaction was more effective in the presence of 0.2 M NaCl or higher. Cellular redox factor, thioredoxin, was able to replace DTT in stimulation of RPA DNA binding activity, suggesting that redox protein may be involved in RPA modulation in vivo. In contrast to wild-type RPA, zinc finger mutant (cysteine to alanine mutation at amino acid 486) did not require DTT for its ssDNA binding activity and is not affected by redox. Together, these results suggest a novel function for a putative zinc finger in the regulation of RPA DNA binding activity through cellular redox.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Structures of heparin-derived tetrasaccharide bound to cobra cardiotoxins: heparin binding at a single protein site with diverse side chain interactions

Cobra cardiotoxins (CTXs) are three-fingered polypeptides with positively charged domains that have been shown to bind to anionic ligands of snake venom citrate, glycosaminoglycans, sulfoglycosphingolipid, and nucleotide triphosphate with various biochemical effects including toxin dimerization, cell surface retention, membrane pore formation, cell internalization and blocking of enzymatic activities of kinase and ATPase. The reported anionic binding sites, however, are found to be different among different CTX homologues for potentially different CTX activities. Herein, by NMR studies of the binding of inorganic phosphate, dATP (stable form of ATP), and heparin-derived tetrasaccharide to Naja atra CTX A1, a novel CTX molecule exhibiting in vivo necrotic activity on skeletal muscle, we demonstrate that diverse ligands binding to CTXs could also occur at a single protein site with flexible side chain interactions. The flexibility of such an interaction is also illustrated by the available heparin-CTX A3 complex structures with different heparin chain lengths binding at the same site. Our results provide a likely structural explanation on how the interaction between heparan sufate and proteins depends more on the overall charge cluster organization rather than on their fine structures. We also suggest that the ligand binding site of CTX homologues can be fine-tuned by nonconserved residues near the binding pocket because of their flexible side chain interaction and dimerization ability, even for the rigid CTX molecules tightened by four disulfide bonds.     

Copper and zinc promote interactions between membrane-anchored peptides of the metal binding domain of the prion protein

The prion protein (PrP) has been identified as a metalloprotein capable of binding multiple copper ions and possibly zinc. Recent studies now indicate that prion self-recognition may be an important factor in both the normal function and misfunction of this protein. We have developed fluorescently labeled models of the prion protein that allow prion-prion interactions and metal binding to be investigated on the molecular level. Peptides encompassing the full metal binding region were anchored to the surface of small unilamellar vesicles, and PrP-PrP interactions were monitored by fluorescence spectroscopy as a function of added metal. Both Cu2+ and Zn2+ were found to cause an increase in the level of PrP-PrP interactions, by 117 and 300%, respectively, whereas other metals such as Ni2+, Co2+, and Ca2+ had no effect. The binding of either of these cofactors appears to act as a switch that induces PrP-PrP interactions in a reversible manner. Both glutamine and tryptophan residues, which occur frequently in the metal binding region of PrP, were found to be important in mediating PrP-PrP interactions. Experiments demonstrate that tryptophan residues are also responsible for the low level of PrP-PrP interactions observed in the absence of Cu2+ and Zn2+, and this is further supported by molecular modeling. Overall, our results indicate that PrP may be a bifunctional molecule capable of responding to fluctuations in both neuronal Cu2+ and Zn2+ levels.     

Zinc is required for folding and binding of a single zinc finger to DNA

A synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a folded conformation in the presence of zinc. The same peptide in the absence of zinc is not folded in a stable tertiary conformation, as determined by NMR. Binding experiments have shown that the peptide binds non-specifically to DNA only in the presence of zinc. Moreover, competitive DNA binding experiments indicate interaction with 3.9 +/- 0.4 base pairs.     

Molecular architecture and domain arrangement of the placental malaria protein VAR2CSA suggests a model for carbohydrate binding

VAR2CSA is the placental-malaria–specific member of the antigenically variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria.