The alpha-helical rod domain of human lamins A and C contains a chromatin binding site

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.     

Lamin A is part of the internal nucleoskeleton of human erythroleukemia cells

Nuclear lamins are the most abundant components of the nuclear lamina, a 10-50-nm-thick fibrous layer underlying the inner nuclear envelope membrane. Nevertheless, a number of recent investigations performed on epithelial and fibroblast cells have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nucleoskeleton. We have studied the subnuclear distribution of lamins A and B1 in human erythroleukemia cells by using immunoblotting analysis and immunofluorescent staining of fractionated nuclei. In intact cells and isolated nuclei, antibodies to lamins A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. However, when chromatin was removed by nuclease digestion and extraction with nonionic detergent or solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix or scaffold. Preferential localization of lamin A to the inner part of the nucleus was also demonstrated by the presence of the majority of lamin A in the solubilized inner nuclear network subfraction. In contrast, lamin B1 was mainly recovered in the fraction corresponding to the nuclear periphery. Double labeling experiments showed that lamin A, but not lamin B1, colocalized with coiled and GATA-1 bodies. Thus, our results support the hypothesis that lamin A, but not lamin B1, may be a component of an internal nucleoskeleton in human erythroleukemia cells.     

The E1B 19K protein associates with lamins in vivo and its proper localization is required for inhibition of apoptosis

Expression of the E1B 19K protein is required to inhibit apoptosis induced by E1A during adenovirus infection and transformation. E1B 19K is homologous to Bcl-2 in function and the two proteins also share limited amino acid sequence homology. Consequently, the E1B 19K and Bcl-2 proteins bind to and inhibit the cellular death-inducing proteins Bax, Bak and Nbk/Bik. Both E1B 19K and Bcl-2 localize to membranes of the nucleus and the endoplasmic reticulum. In addition to membrane association, and unlike Bcl-2, the E1B 19K protein is found associated with intermediate filament proteins in the cytoplasm and the nuclear lamina and copurifies with the lamins both during infection and transformation. While a membrane targeting domain at the C-terminus of Bcl-2 ensures its proper localization, the mechanism by which the E1B 19K protein localizes is unknown. Not surprisingly, lamin A fragments were cloned from a yeast two-hybrid screen for E1B 19K-interacting proteins. The interaction was demonstrated in yeast and mammalian cells in vivo and in vitro and was unique and specific to E1B 19K, with no interaction evident between Bcl-2 and lamin A. Mutants of lamin A/C which localized inappropriately in the cytoplasm or nucleus but retained E1B 19K binding, interfered with the nuclear envelope and cytoplasmic membrane targeting of the E1B 19K protein. Improper localization impaired the ability of the E1B 19K protein to inhibit apoptosis. Thus, proper localization of the E1B 19K protein is required for its function and the interaction of the E1B 19K protein with lamin A/C may represent a means for nuclear envelope localization.     

Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.     

A- and B-type lamins are differentially expressed in normal human tissues

A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies.     

Identification of protein phosphatase 1 as a mitotic lamin phosphatase

At the onset of mitosis, the nuclear lamins are hyperphosphorylated leading to nuclear lamina disassembly, a process required for nuclear envelope breakdown and entry into mitosis. Multiple lamin kinases have been identified, including protein kinase C, that mediate mitotic lamin phosphorylation and mitotic nuclear lamina disassembly. Conversely, lamin dephosphorylation is required for nuclear lamina reassembly at the completion of mitosis. However, the protein phosphatase(s) responsible for the removal of mitotic phosphates from the lamins is unknown. In this study, we use human lamin B phosphorylated at mitosis-specific sites as a substrate to identify and characterize a lamin phosphatase activity from mitotic human cells. Several lines of evidence demonstrate that the mitotic lamin phosphatase corresponds to type 1 protein phosphatase (PP1). First, mitotic lamin phosphatase activity is inhibited by high nanomolar concentrations of okadaic acid and the specific PP1 peptide inhibitor, inhibitor-2. Second, mitotic lamin phosphatase activity cofractionates with PP1 after ion exchange chromatography. Third, microcystin-agarose depletes mitotic extracts of both PP1 and lamin phosphatase activity. Our results demonstrate that PP1 is the major mitotic lamin phosphatase responsible for removal of mitotic phosphates from lamin B, a process required for nuclear lamina reassembly.     

Purification of intact nuclear lamina and identification of novel laminlike proteins in Raji, a cell line devoid of lamins A and C

Research on the structure of the nuclear lamina and the nuclear matrix of cells devoid of lamins A and C has been hampered by the fact that intact residual nuclear structures are difficult to isolate from such cells. In this paper, we show that some extraction parameters, such as buffer composition and the nature of the detergent used to remove nuclear membranes, are critical for achieving isolation of whole nuclear residual structures from the lymphoblastic cell line Raji, used as a model for cells without lamins A and C. Electron microscopic analysis shows that the nuclear lamina of Raji cells is formed by a network of intermediate-size filaments interrupted with circular discontinuities. Both lamins B1 and B2, and lamin D/E, are present in this structure. In addition, a group of 45-kDa proteins or intermediate filament protein--reacting proteins (IFA-RPs), located uniquely in the lamina, were found to exhibit the same immunological and chemical characteristics as lamins. Although they behave like nuclear lamins, microsequencing analysis of the IFA-RPs has revealed no homology with known lamins. These IFA-RPs may contribute to the formation of the nuclear lamina filament network in the absence of lamins A and C.     

Molecular phylogeny of metazoan intermediate filament proteins

We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia.     

Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI

Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with factor XIIa.     

Endogenous asymmetrical dimethylarginine and hypertension associated with puromycin nephrosis in the rat

LBR (lamin B receptor) is an integral protein of the inner nuclear membrane encoded by a gene on human chromosome 1q42.1. LBR has a nucleoplasmic, amino-terminal domain of approximately 200 amino acids followed by a carboxyl-terminal domain similar in sequence to yeast and plant sterol reductases. We have determined the primary structures of two human proteins with strong sequence similarity to the carboxyl-terminal domain of LBR and sterol reductases. Their genes have recently been assigned the symbols TM7SF2 and DHCR7. TM7SF2 mRNA is most predominantly expressed in heart and DHCR7 mRNA mostly in liver and brain. Whereas LBR is localized to the inner nuclear membrane, these two related proteins are in the endoplasmic reticulum. The TM7SF2 gene contains 10 coding exons, and its intron positions are exactly conserved in the part of the LBR gene encoding its carboxyl-terminal domain. Intron positions in the DHCR7 gene are also similar. Both of these new LBR-like genes are on chromosome 11q13. These results describe a human gene family encoding proteins of the inner nuclear membrane and endoplasmic reticulum that function in nuclear organization and/or sterol metabolism.     

Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR

The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy. Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II. To facilitate and confirm NH, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths. Ninety-eight percent of the 1H, 15N, and 13C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments. The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms. The structure is ellipsoidal in shape, approximately 78 A long and 32 A wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha-domain (residues 33-143) consisting of four helices. The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N- and C-terminal domains of intact enzyme I. A comparison with the recently solved X-ray structure of EIN [Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B. R., Peterkofsky, A., & Davies, D. R. (1996) Structure 4, 861-872] indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates. The active site His189 is located in a cleft at the junction of the alpha and alpha/beta domains and has a pKa of approximately 6.3. His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168. Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation.     

Regions of the haptoglobin 5' flanking gene sequence show different binding affinities to nuclear matrix proteins during the acute phase response

The effect of the acute phase response on the affinity of binding between nuclear matrix proteins and the rat haptoglobin (Hp) gene region was examined. Nuclear matrices isolated from acute phase livers were enriched with the 5' Hp gene flanking region (-705/+159), but not with the spliced, protein-coding sequence. Reassociation experiments with isolated nuclear protein matrix spheres and end-labeled fragments I (-146/+156), II (-146/-541), and III (-541/-705) revealed that the matrix proteins displayed an increased binding potential during the acute phase response for all of the examined regions, this being most pronounced for fragment II. BAL 31 digestion of fragment II showed that the sequence element that was responsible for the increased association with nuclear matrix proteins during the acute phase response was a tract of 38 adenine bases. The DNA region established stable associations with nuclear lamin B (67 kDa, pI 5.7) in the controls, and with lamins A (69 kDa, pI 7.0), B, isoforms of lamin C (62 kDa, pI 6.55-6.95), and a 55-kDa (pI 5.9) polypeptide during the acute phase response. Sequence ABC (-165/-56), which overlaps fragments I and II and represents the Hp cis-acting element, did not bind to the non-histone nuclear matrix proteins.     

Identification of the substrate and pseudosubstrate binding sites of phosphorylase kinase gamma-subunit

Using site-directed mutagenesis, we proposed that an autoinhibitory domain(s) is located at the C-terminal region (301-386) of the phosphorylase kinase gamma-subunit (Huang, C.-Y.F., Yuan C.-J., Livanova, N.B., and Graves, D.J. (1993) Mol. Cell. Biochem. 127/128, 7-18). Removal of the putative inhibitory domain(s) by truncation results in the generation of a constitutively active and calmodulin-independent form, gamma 1-300. To probe the structural basis of autoinhibition of gamma-subunit activity, two synthetic peptides, PhK13 (gamma 303-327) and PhK5 (gamma 343-367), corresponding to the two calmodulin-binding regions, were assayed for their ability to inhibit gamma 1-300. Competitive inhibition of gamma 1-300 by PhK13 was found versus phosphorylase b (Ki = 1.8 microM) and noncompetitive inhibition versus ATP. PhK5 showed noncompetitive inhibition with respect to both phosphorylase b and ATP. Calmodulin released the inhibition caused by both peptides. These results indicate that there are two distinct auto-inhibitory domains within the C terminus of the gamma-subunit and that these two domains overlap with the calmodulin-binding regions. Two mutant forms of gamma 1-300, E111K and E154R, were used to probe the enzyme-substrate-binding region using peptide substrate analogs corresponding to residues 9-18 of phosphorylase b (KRK11Q12ISVRGL). The data suggest that Glu111 interacts with the P-3 position of the substrate (Lys11) and Glu154 interacts with the P-2 site (Gln12). Both E111K and E154R were competitively inhibited with respect to phosphorylase b by PhK13, with 14- and 8-fold higher Ki values, respectively, than that observed with the wild-type enzyme. These data are consistent with a model for the regulation of the gamma-subunit of phosphorylase kinase in which PhK13 acts as a competitive pseudosubstrate that directly binds the substrate binding site of the gamma-subunit (Glu111 and Glu154).     

Topology of T cell receptor-peptide/class I MHC interaction defined by charge reversal complementation and functional analysis

The molecular interactions between the CD8 co-receptor dependent N15 and N26 T cell receptors (TCRs) and their common ligand, the vesicular stomatitis virus octapeptide (VSV8) bound to H-2Kb, were studied to define the docking orientation(s) of MHC class I restricted TCRs during immune recognition. Guided by the molecular surfaces of the crystallographically defined peptide/MHC and modeled TCRs, a series of mutations in exposed residues likely contacting the TCR ligand were analyzed for their ability to alter peptide-triggered IL-2 production in T cell transfectants. Critical residues which diminished antigen recognition by 1000 to 10,000-fold in molar terms were identified in both N15 Valpha (alphaE94A or alphaE94R, Y98A and K99) and Vbeta (betaR96A, betaW97A and betaD99A) CDR3 loops. Mutational analysis indicated that the Rp1 residue of VSV8 is critical for antigen recognition of N15 TCR, but R62 of H-2Kb is less critical. More importantly, the alphaE94R mutant could be fully complemented by a reciprocal charge reversal at Kb R62 (R62E). This result suggests a direct interaction between N15 TCR Valpha E94R and Kb R62E residues. As Rp1 of VSV8 is adjacent to R62 in the VSV8/Kb complex and essential for T cell activation, this orientation implies that the N15 Valpha CDR3 loop interacts with the N-terminal residues of VSV8 with the Valpha domain docking to the Kb alpha2 helix while the N15 Vbeta CDR3 loop interacts with the more C-terminal peptide residues and the Vbeta domain overlies the Kb alpha1 helix. An equivalent orientation is suggested for N26, a second VSV8/Kb specific TCR. Given that genetic analysis of two different class II MHC-restricted TCRs and two crystallographic studies of class I restricted TCRs offers a similar overall orientation of V domains relative to alpha-helices, these data raise the possibility of a common docking mode between TCRs and their ligands regardless of MHC restriction.     

Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.     

Synaptotagmin-syntaxin interaction: the C2 domain as a Ca2+-dependent electrostatic switch

Synaptotagmin I is a synaptic vesicle protein that is thought to act as a Ca2+ sensor in neurotransmitter release. The first C2 domain of synaptotagmin I (C2A domain) contains a bipartite Ca2+-binding motif and interacts in a Ca2+-dependent manner with syntaxin, a central component of the membrane fusion complex. Analysis by nuclear magnetic resonance spectroscopy and site-directed mutagenesis shows that this interaction is mediated by the cooperative action of basic residues surrounding the Ca2+-binding sites of the C2A domain and is driven by a change in the electrostatic potential of the C2A domain induced by Ca2+ binding. A model is proposed whereby synaptotagmin acts as an electrostatic switch in Ca2+-triggered synaptic vesicle exocytosis, promoting a structural rearrangement in the fusion machinery that is effected by its interaction with syntaxin.     

The domain structure of human receptor-associated protein. Protease sensitivity and guanidine HCl denaturation

The 39-kDa receptor-associated protein (RAP), a specialized chaperone for endocytic receptors of the low density lipoprotein receptor gene family, is a triplicate repeat sequence (residues 1-100, 101-200, and 201-323, respectively), with the three repeats having different functional roles. The goal of the present study was to use a combination of protease sensitivity and guanidine denaturation analyses to investigate whether human RAP correspondingly contained multiple structural domains. Protease sensitivity analysis using six proteolytic enzymes of varying specificity showed that RAP has two protease-resistant regions contained within repeat 1 (residues 15-94) and repeat 3 (residues 223-323). Guanidine denaturation analysis showed that RAP has two phases in its denaturation, an early denaturation transition at 0.6 M guanidine HCl, and a broad second transition between 1.0 and 3.0 M guanidine HCl. Analysis of the denaturation of the individual repeats showed that, despite the similarity in sequence and protease sensitivity between repeats 1 and 3, repeat 1 was a stable structure, with a sharp transition midpoint at 2.4 M guanidine HCl, while repeat 3 was relatively unstable, with a transition midpoint at 0.6 M guanidine HCl. Repeat 2 had a denaturation profile almost identical to that of repeat 3. Denaturation analysis of the contiguous repeats 1 and 2 (residues 1-210) indicated that repeats 1 and 2 probably interact to form one structural domain represented by the broad transition, while repeat 3 constitutes a separate domain represented by the early transition. A two-domain model of RAP three-dimensional structure is proposed that integrates both structural and functional information, in which a helical segment from repeat 2 interacts with the known three-helix bundle of repeat 1 to form a four-helix bundle structural domain, while repeat 3 forms the other structural domain.     

Topology and functional domains of the yeast pore membrane protein Pom152p

Integral membrane proteins associated with the nuclear pore complex (NPC) are likely to play an important role in the biogenesis of this structure. Here we have examined the functional roles of domains of the yeast pore membrane protein Pom152p in establishing its topology and its interactions with other NPC proteins. The topology of Pom152p was evaluated by alkaline extraction, protease protection, and endoglycosidase H sensitivity assays. The results of these experiments suggest that Pom152p contains a single transmembrane segment with its N terminus (amino acid residues 1-175) extending into the nuclear pore and its C terminus (amino acid residues 196-1337) positioned in the lumen of the nuclear envelope. The functional role of these different domains was investigated in mutants that are dependent on Pom152p for viability. The requirement for Pom152p in strains containing mutations allelic to the NPC protein genes NIC96 and NUP59 could be alleviated by Pom152p's N terminus, independent of its integration into the membrane. However, complementation of a mutation in NUP170 required both the N terminus and the transmembrane segment. Furthermore, mutations in NUP188 were rescued only by full-length Pom152p, suggesting that the lumenal structures play an important role in the function of pore-side NPC structures.     

Crystal structure of the spliceosomal U2B"-U2A' protein complex bound to a fragment of U2 small nuclear RNA

We have determined the crystal structure at 2.4 A resolution of a ternary complex between the spliceosomal U2B"/U2A' protein complex and hairpin-loop IV of U2 small nuclear RNA. Unlike its close homologue the U1A protein, U2B" binds to its cognate RNA only in the presence of U2A', which contains leucine-rich repeats in its sequence. The concave surface of a parallel beta-sheet within the leucine-rich-repeat region of U2A' interacts with the ribonucleoprotein domain of U2B" on the surface opposite its RNA-binding surface. The basic carboxy-terminal region of U2A' interacts with the RNA stem. The crystal structure reveals how protein-protein interaction regulates RNA-binding specificity, and how replacing only a few key residues allows the U2B" and U1A proteins to discriminate between their cognate RNA hairpins by forming alternative networks of interactions.