Ab initio simulations of Cu binding sites on the N-terminal region of prion protein

The human prion protein binds Cu²⁺ ions in the octarepeat domain of the N-terminal tail up to full occupancy at pH 7.4. Recent experiments have shown that the HGGG octarepeat subdomain is responsible for holding the metal bound in a square-planar configuration. By using first principle ab initio molecular dynamics simulations of the Car–Parrinello type, the coordination of copper to the binding sites of the prion protein octarepeat region is investigated. Simulations are carried out for a number of structured binding sites. Results for the complexes Cu(HGGGW)(wat), Cu(HGGG), and [Cu(HGGG)]₂ are presented. While the presence of a Trp residue and a water molecule does not seem to affect the nature of the copper coordination, high stability of the bond between copper and the amide nitrogen of deprotonated Gly residues is confirmed in all cases. For the more interesting [Cu(HGGG)]₂ complex, a dynamically entangled arrangement of the two domains with exchange of amide nitrogen bonds between the two copper centers emerges, which is consistent with the short Cu–Cu distance observed in experiments at full copper occupancy.     

Deconvoluting the Cu2+ binding modes of full-length prion protein

The prion protein (PrP) is a cell-surface Cu(2+)-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23-231) and constructs in which the octarepeat region has been removed, or His(95) and His(110) is replaced by alanine residues, have been used to elucidate the order and mode of Cu(2+) coordination to PrP-(23-231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu(2+) coordination to full-length PrP. At physiological pH, Cu(2+) initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His(95) and His(110). Only subsequent Cu(2+) ions bind to single histidine residues within the octarepeat region. Ni(2+) ions are used to further probe metal binding and, like Cu(2+), Ni(2+) will bind individually to His(95) and His(110), involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu(2+) bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu(2+) binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.     

The configuration of the Cu2+ binding region in full-length human prion protein

The cellular prion protein (PrP^C) is a Cu²⁺ binding protein connected to the outer cell membrane. The molecular features of the Cu²⁺ binding sites have been investigated and characterized by spectroscopic experiments on PrP^C-derived peptides and the recombinant human full-length PrP^C (hPrP-[23-231]). The hPrP-[23-231] was loaded with ⁶³Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP^C/Cu²⁺-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu²⁺ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu²⁺ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP^C. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

EPR of Cu+2 binding to apo-yeast enolase

We have studied the electron paramagnetic resonance (epr) spectra of complexes of apo-yeast enolase with 65Cu+2 in the presence and absence of substrate and magnesium ion. An unusual epr spectrum with large g parallel, large g and A rhombicity and very narrow line-widths (10 G) is seen for the first two 65Cu+2 bound in the presence of substrate 2-phosphoglycerate (2PGA). the epr parameters, consistent with rhombic and tetragonal distortion of an octahedral geometry of the coordination sphere of the Cu+2 are g = (2.123, 2.042, 2.405) and A = (2.58, 4.19, 12.0) mK. The high g parallel and absence of super-hyperfine splitting are strong evidence for absence of nitrogen ligands. In the presence of Mg+2 and 2PGA, the Cu+2-enolase solutions exhibit a complex epr spectrum reflecting exchange and dipolar interaction between the first two Cu+2 ions bound. The spectra of Cu+2 plus enolase in the presence and absence of Mg+2 without 2PGA are distinct but not unambiguous, each reflecting at least two inequivalent binding sites. In addition to providing information on the geometry and location of the divalent cation binding sites, the data show unequivocally that imidazole residues, previously found to have a role in catalysis, do not participate in Cu+2 binding. Although Cu+2 does not activate the enzyme, direct binding measurements show that Cu+2 competes stoichiometrically with the activating ion, Mg+2. A reinterpretation of earlier Mn+2 enolase studies is proposed to reconcile the Cu+2 and Mn+2 data.     

Molecular features of the copper binding sites in the octarepeat domain of the prion protein

Recent evidence suggests that the prion protein (PrP) is a copper binding protein. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds Cu2+ in vivo. In a previous study using peptide design, EPR, and CD spectroscopy, we showed that the HGGGW segment within each octarepeat comprises the fundamental Cu2+ binding unit [Aronoff-Spencer et al. (2000) Biochemistry 40, 13760-13771]. Here we present the first atomic resolution view of the copper binding site within an octarepeat. The crystal structure of HGGGW in a complex with Cu2+ reveals equatorial coordination by the histidine imidazole, two deprotonated glycine amides, and a glycine carbonyl, along with an axial water bridging to the Trp indole. Companion S-band EPR, X-band ESEEM, and HYSCORE experiments performed on a library of 15N-labeled peptides indicate that the structure of the copper binding site in HGGGW and PHGGGWGQ in solution is consistent with that of the crystal structure. Moreover, EPR performed on PrP(23-28, 57-91) and an 15N-labeled analogue demonstrates that the identified structure is maintained in the full PrP octarepeat domain. It has been shown that copper stimulates PrP endocytosis. The identified Gly-Cu linkage is unstable below pH approximately 6.5 and thus suggests a pH-dependent molecular mechanism by which PrP detects Cu2+ in the extracellular matrix or releases PrP-bound Cu2+ within the endosome. The structure also reveals an unusual complementary interaction between copper-structured HGGGW units that may facilitate molecular recognition between prion proteins, thereby suggesting a mechanism for transmembrane signaling and perhaps conversion to the pathogenic form.     

Identification of hyaluronic acid binding sites in the extracellular domain of CD44

CD44 is a polymorphic glycoprotein expressed on the surface of many tissues and cell lines which has been implicated in a number of cellular functions including lymphocyte homing to mucosal lymphoid tissue (Peyers patches), leukocyte activation, lymphopoiesis, and tumor metastasis. The predominant isoform found on human leukocytes, CD44H, is a receptor for hyaluronic acid. Because of the prominent role CD44 plays in diverse biological processes, we set out to identify the hyaluronic acid binding site(s) in the extracellular domain of CD44H. Using truncation and site-directed mutagenesis we identified two regions containing clusters of conserved basic residues which are important in hyaluronic acid binding. One of these regions is situated near the NH2 terminus and is homologous to other hyaluronic acid binding proteins including cartilage link protein. The other more membrane proximal region lies outside the link protein homologous domain. Mutagenesis of basic residues within these regions established their role as determinants in hyaluronic acid binding. Mutation of Arg 41, a position where a basic residue is conserved in all hyaluronic acid binding proteins, completely abolished binding suggesting that this residue plays a critical role in hyaluronic acid binding.     

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.     

Antagonistic roles of the N-terminal domain of prion protein to doppel

Prion protein (PrP)-like molecule, doppel (Dpl), is neurotoxic in mice, causing Purkinje cell degeneration. In contrast, PrP antagonizes Dpl in trans, rescuing mice from Purkinje cell death. We have previously shown that PrP with deletion of the N-terminal residues 23–88 failed to neutralize Dpl in mice, indicating that the N-terminal region, particularly that including residues 23–88, may have trans-protective activity against Dpl. Interestingly, PrP with deletion elongated to residues 121 or 134 in the N-terminal region was shown to be similarly neurotoxic to Dpl, indicating that the PrP C-terminal region may have toxicity which is normally prevented by the N-terminal domain in cis. We recently investigated further roles for the N-terminal region of PrP in antagonistic interactions with Dpl by producing three different types of transgenic mice. These mice expressed PrP with deletion of residues 25–50 or 51–90, or a fusion protein of the N-terminal region of PrP with Dpl. Here, we discuss a possible model for the antagonistic interaction between PrP and Dpl.Key words: prion protein, doppel, neurotoxic signal, neurodegeneration, neuroprotection, prion diseaseThe normal prion protein, termed PrP^C, is a membrane glycoprotein tethered to the outer cell surface via a glycosylphosphatidylinositol (GPI) anchor moiety.^1,2 It is ubiquitously expressed in neuronal and non-neuronal tissues, with highest expression in the central nervous system, particularly in neurons.³ The physiological function of PrP^C remains elusive. We and others have shown that PrP^C functionally antagonizes doppel (Dpl), a PrP-like GPI-anchored protein with ∼23% identity in amino acid composition to PrP, protecting Dpl-induced neurotoxicity in mice.^4–7 Dpl is encoded on Prnd located downstream of the PrP gene (Prnp) and expressed in the testis, heart, kidney and spleen of wild-type mice but not in the brain where PrP^C is actively expressed.^4,5,8 However, when ectopically expressed in brains, particularly in cerebellar Purkinje cells, Dpl exerts a neurotoxic activity, causing ataxia and Purkinje cell degeneration in Ngsk, Rcm0 and Zrch II lines of mice devoid of PrP^C (Prnp^0/0).^4,9,10 In these mice, Dpl was abnormally controlled by the upstream Prnp promoter.^4,5 This is due to targeted deletion of part of Prnp including a splicing acceptor of exon 3.¹¹ Pre-mRNA starting from the residual exon1/2 of Prnp was abnormally elongated until the end of Prnd and then intergenically spliced between the residual Prnp exons 1/2 and the Prnd coding exons.^4,5 As a result, Dpl was ectopically expressed under the control of the Prnp promoter in the brain, particularly in neurons including Purkinje cells.^4,5 In contrast, in other Prnp^0/0 lines, such as Zrch I and Npu, the splicing acceptor was intact, resulting in normal Purkinje cells without ectopic expression of Dpl in the brain.⁴The molecular mechanism of the antagonistic interaction between PrP^C and Dpl remains unknown. We recently showed that the N-terminal half of PrP^C includes elements that might mediate cis or trans protection against Dpl in mice, ameliorating Purkinje cell degeneration.¹² We also showed that the octapeptide repeat (OR) region in the N-terminal domain is dispensable for PrP^C to neutralize Dpl neurotoxicity in mice.¹² Here, possible molecular mechanisms for the antagonism between PrP^C and Dpl will be discussed.     

Antagonistic roles of the N-terminal domain of prion protein to doppel

Prion protein (PrP)-like molecule, doppel (Dpl), is neurotoxic in mice, causing Purkinje cell degeneration. In contrast, PrP antagonizes Dpl in trans, rescuing mice from Purkinje cell death. We have previously shown that PrP with deletion of the N-terminal residues 23-88 failed to neutralize Dpl in mice, indicating that the N-terminal region, particularly that including residues 23-88, may have trans-protective activity against Dpl. Interestingly, PrP with deletion elongated to residues 121 or 134 in the N-terminal region was shown to be similarly neurotoxic to Dpl, indicating that the PrP C-terminal region may have toxicity which is normally prevented by the N-terminal domain in cis. We recently investigated further roles for the N-terminal region of PrP in antagonistic interactions with Dpl by producing three different types of transgenic mice. These mice expressed PrP with deletion of residues 25-50 or 51-90, or a fusion protein of the N-terminal region of PrP with Dpl. Here, we discuss a possible model for the antagonistic interaction between PrP and Dpl .     

Localization of the N-terminal domain in light-harvesting chlorophyll a/b protein by EPR measurements

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.     

Influence of the N-terminal domain on the aggregation properties of the prion protein

Prion diseases appear to be caused by the aggregation of the cellular prion protein (PrP(C)) into an infectious form denoted PrP(Sc). The in vitro aggregation of the prion protein has been extensively investigated, yet many of these studies utilize truncated polypeptides. Because the C-terminal portion of PrP(Sc) is protease-resistant and retains infectivity, it is assumed that studies on this fragment are most relevant. The full-length protein can be distinguished from the truncated protein because it contains a largely structured, alpha-helical, C-terminal region in addition to an N terminus that is unstructured in the absence of metal ion binding. Herein, the in vitro aggregation of a truncated portion of the prion protein (PrP 90-231) and a full-length version (PrP 23-231) were compared. In each case, concentration-dependent aggregation was analyzed to discern whether it proceeds by a nucleation-dependent pathway. Both protein constructs appear to aggregate via a nucleated polymerization with a small nucleus size, yet the later steps differ. The full-length protein forms larger aggregates than the truncated protein, indicating that the N terminus may mediate higher-order aggregation processes. In addition, the N terminus has an influence on the assembly state of PrP before aggregation begins, causing the full-length protein to adopt several oligomeric forms in a neutral pH buffer. Our results emphasize the importance of studying the full-length protein in addition to the truncated forms for in vitro aggregation studies in order to make valid hypotheses about the mechanisms of prion aggregation and the distribution of aggregates in vivo.     

Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies

Human Chk2 is a newly identified tumor suppressor protein involved in signaling pathways in response to DNA damage. The protein consists of a forkhead-associated (FHA) domain and a kinase domain. Identification of binding partners of the Chk2FHA domain is important in understanding the roles of Chk2 in signaling. We report development of an approach involving the use of combinatorial libraries, pull-down assays, surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) methods to identify possible candidates for the binding sites of Chk2FHA. The approach has been used to identify Thr329 of p53 and Thr1852 of breast cancer type 1 susceptibility protein (BRCA1) as very likely biological binding sites of Chk2FHA. The results provide useful leads for further biological analyses of cell signaling involving the FHA domain of Chk2 protein.     

Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A

Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.     

Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.     

羊朊毒体单抗结合表位分析

通过分段表达PrP核心片段和人工合成多肽,分析5株羊朊毒体单抗结合表位。分段表达PrP核心片段,通过PCR方法扩增目的片段,经酶切、连接后,将目的片段插入质粒pET32a,在大肠杆菌BL21中表达。将表达的系列融合蛋白与单抗进行免疫转印试验,根据反应情况确定单抗结合的大致部位,在此基础上设计合成多条针对性多肽,用ELISA方法进一步确定3株单抗的结合部位;通过与6段融合蛋白反应证明5株单抗的结合部位分别为:2H3在199aa～213aa之间,4C6、5F11和7F11在139aa～168aa之间,7F1在214aa～227aa之间,与3段人工合成多肽进行ELISA反应进一步得到4C6、5F11和7F11抗原结合表位在149aa～158aa之间;本研究确定了5株单抗在PrP分子上的结合部位,为羊痒病和牛海绵状脑病的检测、发病机制的研究奠定了基础。     

Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis

The newly discovered laminin alpha(5) chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha(5) N-terminal globular domain VI (approximately 270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, (123)GQVFHVAYVLIKF(135) and S6, (225)RDFTKATNIRLRFLR(239), within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr(130), Arg(225), Lys(229), and Arg(239)) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg(239) of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha(3)beta(1) was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha(5) chain N-terminal globular domain VI are critical for cell surface receptor binding.     

Identification of microtubule binding sites in the Ncd tail domain

Nonclaret disjunctional (Ncd) is a minus end-directed, C-terminal motor protein that is required for spindle assembly and maintenance during meiosis and early mitosis in Drosophila oocytes and early embryos. Ncd has an ATP-independent MT binding site in the N-terminal tail domain, and an ATP-dependent MT binding site in the C-terminal motor domain. The ability of Ncd to cross-link MTs through the action of these binding sites may be important for Ncd function in vivo. To identify the region(s) responsible for ATP-independent MT interactions of Ncd, 12 cDNAs coding various regions of Ncd tail domain were expressed in E. coli as C-terminal fusions to thioredoxin (Trx). Ncd tail fusion proteins (TrxNT) were purified by ion exchange (S-Sepharose) and/or Talon metal affinity chromatography. Purified TrxNT and NT proteins were analyzed in microtubule (MT) cosedimentation and bundling assays to identify which tail proteins were able to bind and bundle MTs. Based on the results of these experiments, all TrxNT and NT proteins that showed MT binding activity also bundled MTs, and there are two ATP-independent MT interaction sites in the tail region: one within amino acids 83-100 that exhibits conformation-independent, high-affinity MT binding activity; and another within amino acids 115-187 that exhibits conformation-dependent, lower affinity MT binding activity. It is possible that both of these MT interacting sites combine in the native protein to form a single MT binding site that allows the Ncd tail to bind cargo MTs in vivo.     

Dynamics in the Mn2+ binding site in single crystals of concanavalin A revealed by high-field EPR spectroscopy

EPR spectroscopy at 95 GHz was used to characterize the dynamics at the Mn(2+) binding site in single crystals of the saccharide-binding protein concanavalin A. The zero-field splitting (ZFS) tensor of the Mn(2+) was determined from rotation patterns in the a-c and a-b crystallographic planes, acquired at room temperature and 4.5 K. The analysis of the rotation patterns showed that while at room temperature there is only one type of Mn(2+) site, at low temperatures two types of Mn(2+) sites, not related by any symmetry, are distinguished. The sites differ in the ZFS parameters D and E and in the orientation of the ZFS tensor with respect to the crystallographic axes. Temperature-dependent EPR measurements on a crystal oriented with its crystallographic a axis parallel to the magnetic field showed that as the temperature increases, the two well-resolved Mn(2+) sextets gradually coalesce into a single sextet at room temperature. The line shape changes are characteristic of a two-site exchange. This was confirmed by simulations which gave rates in the range of 10(7)-10(8) s(-1) for the temperature range of 200-266 K and an activation energy of 23.8 kJ/mol. This dynamic process was attributed to a conformational equilibrium within the Mn(2+) binding site which freezes into two conformations at low temperatures.     

Characterization of Cu2+-binding modes in the prion protein by visible circular dichroism and multivariate curve resolution

Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu²⁺ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu²⁺-binding structures to the pure component spectra.