Lysine-dependent multipoint binding of the Borrelia burgdorferi virulence factor outer surface protein E to the C terminus of factor H

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18-20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala(151)-Lys(166)) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.     

Outer surface protein E antibody response and its effect on complement factor H binding to OspE in Lyme borreliosis

Borrelia burgdorferi sensu stricto and B. afzelii, but not B. garinii, are able to escape complement attack by binding factor H via OspE proteins. Recent finding of ospE genes also in B. garinii isolates has raised the question whether, under in vivo-conditions, B. garinii also expresses OspE proteins and consequently induces an antibody response. We set up an IgG ELISA by using recombinant OspE as an antigen. Sixty percent of acute and 64% of convalescent 25 erythema migrans patient samples were positive for anti-OspE antibodies. Anti-OspE antibodies were also found in the sera (83.6%) and cerebrospinal fluids (36%) of patients with neuroborreliosis. Since B. garinii is the major causative agent of neuroborreliosis, the result suggests that OspE is expressed by B. garinii in vivo. Of the 10 acrodermatitis chronica atrophicans patients, 80% had anti-OspE antibodies. Anti-OspE antibody positive sera inhibited factor H binding to Borrelia more efficiently than normal control sera (65% vs. 33.7%). Our results indicate that Borrelia spirochetes, including B. garinii, can induce the production of anti-OspE antibodies. This implies that OspE protein is produced in vivo by B. garinii possibly enabling it to escape complement and cause a CNS infection.     

Complement inhibitor factor H binding to Lyme disease spirochetes is mediated by inducible expression of multiple plasmid-encoded outer surface protein E paralogs

Borrelia burgdorferi spirochetes can circumvent the vertebrate host's immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.     

Accessibility of histone H1(0) and its structural domains to antibody binding in mononucleosomes

This work is devoted to the study of the immunoreactivity of histone H1(0) and its major structural domains in mononucleosomes. Three types of antibody populations were used: (i) anti-H1(0) which reacted with antigenic determinants situated along the whole polypeptide chain; (ii) anti-GH5 which recognized epitopes located in the globular region; and (iii) anti-C-tail antibodies reacting specifically with fragment 99-193 of the protein molecule. The anti-GH5 antibodies gave a weak reaction, the C-tail-specific antibodies reacted relatively strongly and the antiserum to the intact molecule showed an intermediate level of reactivity. The relative intensities of the immunoreaction could be interpreted as reflecting the exposure of the antigenic determinants of the individual protein domains in the monosome particle.     

Roles of H1 domains in determining higher order chromatin structure and H1 location

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.     

Accessibility of histone H1° and its structural domains to antibody binding in extended and folded chromatin

The aim of this work was to study the accessibility of histone H1° and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1° which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1° which recognized epitopes located in the globular region of H1° and (3) anti-C-tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1° did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1° in chromatin was much less than that of histone H1.Abbreviations ELISA Enzyme-Linked Immunosorbent Assay - G-domain Globular domain - IgG Immunoglobulin G - SDS Sodium Dodecylsulphate     

The cytoplasmic tail of NSP4, the endoplasmic reticulum-localized non-structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domains.

The final steps in the assembly of rotavirus occur in the lumen of the endoplasmic reticulum (ER). Targeting of the immature inner capsid particle (ICP) to this compartment is mediated by the cytoplasmic tail of NSP4, a non-structural virus glycoprotein located in the ER membrane. To delineate structural and functional features of NSP4, soluble fragments of the cytoplasmic tail have been expressed and purified. Our analysis combines a functional assay for ICP binding with biochemical and CD spectroscopic studies to examine the secondary and quaternary structure. The ICP-binding domain is located within the C-terminal 20 amino acids of the polypeptide. A second region, distinct from this receptor domain, adopts an alpha-helical coiled coil structure and mediates the oligomerization of the virus binding domains into a homotetramer. The domain organization of the cytoplasmic fragments of NSP4 suggests a novel structure for an icosahedral virus receptor protein in which C-terminal binding sites for immature rotavirus particles are connected to an alpha-helical coiled coil stalk which projects from the ER membrane.     

Expression, purification, and characterization of coiled coil and leucine zipper domains of C-terminal myosin binding subunit of myosin phosphatase for solution NMR studies

Protein-protein interactions between MBS and PKG are mediated by the involvement of C-terminal domain of MBS, MBS(CT180) and N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKG-Iα, PKG-Iα1(-59). MBS(CT180) is comprised of three structurally variant domains of non-CC, CC, and LZ nature. Paucity of three-dimensional structural information of these MBS domains precludes atomic level understanding of MBS-PKG contractile complex structure. Here we present data on cloning, expression, and purification of CC, LZ, and CCLZ domains of MBS(CT180) and their biophysical characterization using size exclusion chromatography (SEC), circular dichroism (CD), and two-dimensional (1)H-(15)N HSQC NMR. The methods as detailed resulted in high level protein expression and high milligram quantities of purified isotopically ((15)N and (13)C) enriched polypeptides. SEC, CD, and (1)H-(15)N HSQC NMR experiments demonstrated that recombinantly expressed MBS CC domain is well folded and exists as a dimer within physiologic pH range, which is supported by our previous findings. The dimerization of CC MBS is likely mediated through formation of coiled coil conformation. In contrast, MBS LZ domain was almost unfolded that exists as non-stable low structured monomer within physiologic pH range. Protein folding and stability of MBS LZ was improved as a function of decrease in pH that adopts a folded, stable, and structured conformation at acidified pH 4.5. SEC and NMR analyses of LZ vs. CCLZ MBS domains indicated that inclusion of CC domain partially improves protein folding of LZ domain.     

Putative coiled-coil structural elements of the BBA68 protein of Lyme disease spirochetes are required for formation of its factor H binding site

Factor H and factor H like-protein 1 (FHL-1) are complement regulatory proteins that serve as cofactors for the factor I-mediated cleavage of C3b. Some Lyme disease and relapsing fever spirochete species bind factor H to their surface to facilitate immune evasion. The Lyme disease spirochetes produce several factor H binding proteins (FHBPs) that form two distinct classes. Class I FHBPs (OspE orthologs and paralogs) bind only factor H, while class II FHBPs (BBA68) bind both factor H and FHL-1. BBA68 belongs to a large paralogous protein family, and of these paralogs, BBA69 is the member most closely related to BBA68. To determine if BBA69 can also bind factor H, recombinant protein was generated and tested for factor H binding. BBA69 did not exhibit factor H binding ability, suggesting that among family 54 paralogs, factor H binding is unique to BBA68. To identify the determinants of BBA68 that are involved in factor H binding, truncation and site-directed mutational analyses were performed. These analyses revealed that the factor H binding site is discontinuous and provide strong evidence that coiled-coil structural elements are involved in the formation of the binding site.     

Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis

The major outer membrane protein (MOMP) of Chlamydia trachomatis carries serovar-, subspecies-, species- and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different serovars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero-taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovar L1 MOMP.     

Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H

Streptococcus pneumoniae, also known as the pneumococcus, contains several surface proteins that along with the polysaccharide capsule function in antiphagocytic activities and evasion of the host immune system. These pneumococcal proteins interact with the host immune system in various ways and possess a wide range of biological activities that suggests that they may be involved at different stages of pneumococcal infection. PspC, also known as CbpA and SpsA, is one of several pneumococcal surface proteins that binds host proteins, including factor H (FH) and secretory IgA (sIgA) via the secretory component. Previous work by our laboratory has demonstrated that PspC on the surface of live pneumococcal cells binds FH. This paper provides evidence that FH activity is maintained in the presence of PspC and that the PspC binding site is located in the short consensus repeat 6-10 region of FH. We also report for the first time that although both FH and sIgA binding has been localized to the alpha-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the alpha-helical region of PspC.     

SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil

The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.     

The relative roles of factor H binding protein, neisserial surface protein A, and lipooligosaccharide sialylation in regulation of the alternative pathway of complement on meningococci

Neisseria meningitidis inhibits the alternative pathway (AP) of complement using diverse mechanisms, including expression of capsule (select serogroups), Neisserial surface protein A (NspA), factor H (fH) binding protein (fHbp), and lipooligosaccharide (LOS) sialylation. The contribution of the latter three molecules in AP regulation in encapsulated meningococci was studied using isogenic mutants. When LOS was unsialylated, deleting NspA alone from group A strain A2594 (low fHbp/high NspA) significantly increased AP-mediated C3 deposition. C3 deposition further increased ～2-fold in a ΔfHbpΔNspA double mutant, indicating cooperative fHbp function. LOS sialylation of A2594 ΔfHbpΔNspA decreased the rate of C3 deposition, revealing AP inhibition by LOS sialic acid. Maximal C3 deposition on group B strain H44/76 (high fHbp/low NspA) occurred when all three molecules were absent; again, LOS sialylation attenuated the AP in the absence of both fHbp and NspA. When H44/76 LOS was unsialylated, both fHbp and NspA independently inhibited the AP. LOS sialylation enhanced binding of fH C-terminal domains 18-20 to C3 fragments deposited on bacteria. Interaction of meningococci with nonhuman complement is relevant for animal models and vaccine evaluation studies that use nonhuman complement. Consistent with their human-specific fH binding, neither fHbp nor NspA regulated the rat AP. However, LOS sialylation inhibited the rat AP and, as with human serum, enhanced binding of rat fH to surface-bound C3. These data highlight the cooperative roles of meningococcal NspA and fHbp in regulating the human AP and broaden the molecular basis for LOS sialylation in AP regulation on meningococci in more than one animal species.     

Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt

The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

The importance of framework residues H6, H7 and H10 in antibody heavy chains: experimental evidence for a new structural subclassification of antibody V(H) domains

The N-terminal segment (FR-H1) of the heavy chain (V(H)) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6(Q)7(P)10(A) and for two of the mutants, the X-ray structures have been determined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first beta-strand of V(H), not observed in natural Fv fragments and forms a novel type of diabody. Two V(H) domains of the mutant associate by swapping the first beta-strand. Concentration-dependent changes in Trp fluorescence indicate that this dimerization also occurs in solution. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries.     

Variable regions in the sushi domains 6-7 and 19-20 of factor H in animals and human lead to change in the affinity to factor H binding protein of Borrelia

Borrelia binds host's complement regulatory factor H (fH) to evade complement attack. However, binding affinities between fH-binding-proteins (FHBPs) of Borrelia and fH from various hosts are disparate. Experiments performed to unfold the underlying molecular basis of this disparity revealed that recombinant BbCRASP-1 (major FHBP of Borrelia burgdorferi) neither interacted with sushi 6-7, nor with sushi 19-20 domains of fH in cattle and pig, however, showed binding affinity to both sushi domains of human fH, sushi 6-7 of mouse and sushi 19-20 of sheep. Further, peptide-spot assay revealed three major binding sites (sushi 6:(335-346), sushi 7:(399-410) and sushi 20:(1205-1227)) in human fH that can form BbCRASP-1:fH interface, while (337)HENMR(341) residues in sushi 6 are crucial for rigid BbCRASP-1:fH complex formation. Amino acid stretches DTIEFTCRYGYRPRTALHTFRTT in ovine sushi 19-20 and SAYWEKVYVQGQ in mouse sushi 7 were important sites for fH:BbCRASP-1 interaction. Comparative analysis of the amino acid sequences of sushi 6 of cattle, pig and human revealed that bovine and porcine fH lack methionine and arginine in HENMR pocket, that may impede formation of fH:BbCRASP-1 interface. Increasing numbers of FHBPs from animal and human pathogens are being discovered, thus results presented here can be important benchmark for study of other FHBPs:FH interactions.     

Cooperativity of the N- and C-terminal domains of insulin-like growth factor (IGF) binding protein 2 in IGF binding

A family of six insulin-like growth factor (IGF) binding proteins (IGFBP-1-6) binds IGF-I and IGF-II with high affinity and thus regulates their bioavailability and biological functions. IGFBPs consist of N- and C-terminal domains, which are highly conserved and cysteine-rich, joined by a variable linker domain. The role of the C-domain in IGF binding is not completely understood in that C-domain fragments have very low or even undetectable IGF binding affinity, but loss of the C-domain dramatically disrupts IGF binding by IGFBPs. We recently reported the solution structure and backbone dynamics of the C-domain of IGFBP-2 (C-BP-2) and identified a pH-dependent heparin binding site [Kuang, Z., Yao, S., Keizer, D. W., Wang, C. C., Bach, L. A., Forbes, B. E., Wallace, J. C., and Norton, R. S. (2006) Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2), J. Mol. Biol. 364, 690-704]. Here, we have analyzed the molecular interactions among the N-domain of IGFBP-2 (N-BP-2), C-BP-2, and IGFs using cross-linking and nuclear magnetic resonance (NMR) spectroscopy. The binding of C-BP-2 to the IGF-I.N-BP-2 binary complex was significantly stronger than the binding of C-BP-2 to IGF-I alone, switching from intermediate exchange to slow exchange on the NMR time scale. A conformational change or stabilization of the IGF-I Phe49-Leu54 region and the Phe49 aromatic ring upon binding to the N-domains, as well as an interdomain interaction between N-BP-2 and C-BP-2 (which is also detectable in the absence of ligand), may contribute to this cooperativity in IGF binding. Glycosaminoglycan binding by IGFBPs can affect their IGF binding although the effects appear to differ among different IGFBPs; here, we found that heparin bound to the IGF-I.N-BP-2.C-BP-2 ternary complex, but did not cause it to dissociate.     

OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively.     

Tpr, a large coiled coil protein whose amino terminus is involved in activation of oncogenic kinases, is localized to the cytoplasmic surface of the nuclear pore complex

From a panel of monoclonal antibodies raised against fractions of rat liver nuclear envelopes (NEs), we have identified an antibody, RL30, which reacts with novel nuclear pore complex (NPC) antigens that are not O-glycosylated. By immunofluorescence staining of cultured cells, RL30 reacts exclusively with the NE in a punctate pattern that largely coincides with that of identified NPC proteins. RL30 labels only the cytoplasmic surface of the NPC in immunogold electron microscopy, predominantly in peripheral regions nearby the cytoplasmic ring. In immunoblots of isolated rat liver NEs and cultured rat cells, RL30 recognizes a 265-kD band, as well as a series of 175-265-kD bands in rat liver NEs that are likely to be proteolytic products of p265. Sequencing of peptides from the 175- and 265-kD RL30 antigens of rat liver revealed that they are both closely related to human Tpr, a protein whose amino-terminal 150-250 amino acids appear in oncogenic fusions with the kinase domains of the met, trk, and raf protooncogenes. We found that in vitro translation of human Tpr mRNA yields a major 265-kD band. Considered together, these data indicate that the 265-kD RL30 antigen in the NPC is the rat homologue of Tpr. Interestingly, Tpr contains an exceptionally long predicted coiled coil domain (approximately 1600 amino acids). The localization and predicted structure of Tpr suggest that it is a component of the cytoplasmic fibrils of the NPC implicated in nuclear protein import. Immunofluorescence microscopy shows that during NPC reassembly at the end of mitosis, Tpr becomes concentrated at the NE significantly later than O-linked glycoproteins, including p62. This indicates that reassembly of the NPC after mitosis is a stepwise process, and that the Tpr-containing peripheral structures are assembled later than p62.