Carboxyl Domain Research Articles

Domain Interactions in the Gelatinase A·TIMP-2·MT1-MMP Activation Complex: THE ECTODOMAIN OF THE 44-kDa FORM OF MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE DOES NOT MODULATE GELATINASE A ACTIVATION

On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly(285)-Cys(508)), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.

Zinc finger and carboxyl regions of adenovirus E1A 13S CR3 are important for transactivation of the cytomegalovirus major immediate early promoter by adenovirus.

Reactivation of latent cytomegalovirus (CMV) is an important cause of disease in susceptible patients. We previously demonstrated that an adenovirus early gene product can transactivate the CMV major immediate early (IE) promoter in inflammatory cells. This effect was due to the conserved region 3 (CR3) of the adenovirus E1A 13S gene product. There are two domains in the CR3 region, a zinc finger (aa 147-177) and a carboxyl (aa 180-188) domain. Both are crucial for transactivation of downstream promoter elements of adenovirus in E1A 13S. We sought to determine if either or both of these specific domains is also necessary for transactivation of the CMV IE promoter by the adenovirus E1A 13S gene product. We cotransfected T-lymphocyte Jurkat cells and monocyte/macrophage-like THP-1 cells with plasmids expressing wild-type (WT) or CR3 mutant E1A 13S and a CMV IE chloramphenicol acetyltransferase (CAT) reporter construct. With extracts of cells coinfected with E1A WT set to 100%, mutation in the zinc finger domain, the carboxyl domain, or both domains decreased CMV IE CAT activity by >/= 96%. In contrast, a mutation in the region between the zinc finger and carboxyl domains reduced CMV IE CAT activity by only 24 to 26%. Mixing studies in Jurkat cells confirmed the importance of these domains. We also evaluated the active site of the CMV IE promoter involved in transactivation in THP-1 cells using CMV IE promoter deletions and single promoter element constructs. These studies showed that progressive deletion of the 19-bp CMV IE repeats containing cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) sites resulted in progressive loss of activity. The importance of this element was confirmed using single promoter elements containing CMV IE 16-, 18-, 19-, and 21-bp repeats. Finally, using a 19-bp single promoter element construct and the CR3 mutants we demonstrated that mutations in the zinc finger (C171S) carboxyl region (S185N) or both regions (C171S/ S185N) resulted in significant (83, 94, and 85%) loss of activity. We conclude that the zinc finger and carboxyl domains of the CR3 region of E1A 13S are necessary for transactivation of the CMV promoter and that this occurs mainly through activation of the 19-bp CREB/ATF site of the promoter.

Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface.

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.

The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3′ genomic RNA

Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3′ non-coding region of IBV RNA. In order to identify domains in the N protein that bind to RNA, the whole protein (409 amino acids) and six overlapping fragments were expressed as fusion polypeptides with six histidine-tags. Using gel shift assays, the intact N protein and amino polypeptides, from residues 1 to 171 and residues 1 to 274, and carboxyl polypeptides, extending from residues 203 to 409 and residues 268 to 407, were found to interact with positive-stranded IBV RNA representing the 3′ end of the genome. The two 32P-labeled probes that interacted with N and the amino and carboxyl fragments of N were RNA consisting of the IBV N gene and adjacent 3′ non-coding terminus, and RNA consisting of the 155-nucleotide sequences at the 3′ end of the 504-nt 3′ untranslated region. In contrast, the polypeptide fragment from the middle region, residues 101–283, did not interact with these 3′ IBV RNAs. The binding site in the amino region of N was either not present or only partially present in the first 91 residues because no interaction with RNA was observed with the polypeptide incorporating these residues. Cache Valley virus N expressed with a histidine tag, bovine serum albumin, and the basic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1–274 fragment needed for equivalent shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain.

Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

Cytoplasmic amino and carboxyl domains form a wide intracellular vestibule in an inwardly rectifying potassium channel.

We have studied the structural components and architecture of the intracellular vestibule of a strongly rectifying channel (Kir2.1) expressed in Xenopus oocytes. Putative vestibule-lining residues were identified by systematically examining covalent modification by sulfhydryl-specific reagents of cysteine residues engineered into two cytoplasmic regions. In a stretch of 33 amino acids in the amino terminus (from C54 to V86) and 22 amino acids in the carboxyl terminus (from R213 to S234), 15 and 11 residues, respectively, were found to be accessible to methanethiosulfonate ethylammonium (MTSEA) or methanethiosulfonate ethyltrimethylammonium (MTSET) and presumably project into the aqueous intracellular vestibule. The pattern of accessibility suggests that both stretches may adopt an extended loop structure. To explore the physical dimension of the intracellular vestibule, we covalently linked a constrained number (one to four) of positively charged moieties of different sizes to the E224 position and found that this vestibule region is sufficiently wide to accommodate four modifying groups with dimensions of 12 A x 10 A x 6 A. These results suggest that regions in both the amino and carboxyl domains of Kir2.1 channel form a long and wide intracellular vestibule that protrudes beyond the membrane into the cytoplasm.

The Arabidopsis blue light receptor cryptochrome 2 is a nuclear protein regulated by a blue light-dependent post-transcriptional mechanism.

Cryptochrome 2 is a flavin-type blue light receptor mediating floral induction in response to photoperiod and a blue light-induced hypocotyl growth inhibition. cry2 is required for the elevated expression of the flowering-time gene CO in response to long-day photoperiods, but the molecular mechanism underlying the function of cry2 is not clear. The carboxyl domain of cry2 bears a basic bipartite nuclear localization signal, and the cry2 protein was co-fractionated with the nucleus. Analysis of transgenic plants expressing a fusion protein of CRY2 and the reporter enzyme GUS (GUS-CRY2) indicated that the GUS-CRY2 fusion protein accumulated in the nucleus of transgenic plants grown in dark or light. The C-terminal domain of cry2 that contains the basic bipartite nuclear localization signal was sufficient to confer nuclear localization of the fusion protein. Phenotypic analysis of transgenic plants expressing the fusion protein GUS-CRY2 demonstrated that GUS-CRY2 acts as a functional photoreceptor in vivo, mediating the blue light-induced inhibition of hypocotyl elongation. These results strongly suggest that cry2 is a nuclear protein. Although no obvious light regulation was found for the nuclear compartmentation of GUS-CRY2 fusion protein, the abundance of GUS-CRY2 was regulated by blue light in a way similar to that of cry2.

Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

Structure, function and regulation of pyruvate carboxylase

Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145-->Ala, Arg451-->Cys, Ala610-->Thr and Met743-->Thr.

Identification of the Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Binding Site on the Hemopexin Carboxyl Domain of Human Gelatinase A by Site-directed Mutagenesis: THE HIERARCHICAL ROLE IN BINDING TIMP-2 OF THE UNIQUE CATIONIC CLUSTERS OF HEMOPEXIN MODULES III AND IV

Cell surface activation of progelatinase A occurs in a quaternary complex with the tissue inhibitor of metalloproteinases-2 (TIMP-2) and two membrane-type matrix metalloproteinases. We have mutated the unique cationic clusters found in hemopexin modules III and IV of the carboxyl domain (C domain) of human gelatinase A to determine their role in binding TIMP-2. Twelve single, double, and triple site-directed mutations were produced that exhibited different TIMP-2 binding properties. Notably, single alanine substitutions at Lys547 and Lys617 reduced TIMP-2 binding by an order of magnitude from that of the recombinant wild-type C domain. Mutations that completely disrupted the C domain.TIMP-2 interaction were K558A/R561A, K610T/K617A, and K566A/K568A/K617A. A triple mutation, K566A/K568A/K575A, having TIMP-2 binding indistinguishable from the wild-type C domain (Kd 3.0 x 10(-8) M), showed that simple reduction of net positive charge does not reduce TIMP-2 affinity. Because the double mutation K566A/K568A also did not alter TIMP-2 binding, these data do not confirm previously reported chimera studies that indicated the importance of the triple lysine cluster at positions 566/567/568 in TIMP-2 binding. Nonetheless, a subtle role in TIMP-2 interaction for the 566/567/568-lysine triad is indicated from the enhanced reduction in TIMP-2 binding that occurs when mutations here were combined with K617A. Thus, these analyses indicate that the TIMP-2 binding surface lies at the junction of hemopexin modules III and IV on the peripheral rim of the gelatinase A C domain. This location implies that considerable molecular movement of the TIMP-2. C domain complex would be needed for the bound TIMP-2 to inhibit in cis the gelatinase A active site.

CDNA Cloning and Characterization of Sciellin, a LIM Domain Protein of the Keratinocyte Cornified Envelope

Sciellin is a precursor of the cornified envelopes of mammalian keratinizing tissues. We have cloned the cDNA encoding sciellin by screening a human keratinocyte expression library with a sciellin-specific monoclonal antibody. The composite cDNA of 2.35 kilobase pairs encodes a protein of 75.3 kDa with a pI of 10.09. The translated sequence has a central domain containing 16 repeats of 20 amino acids each that is rich in Gln and Lys residues, which are potential transglutaminase substrates, and a carboxyl domain, which contains a single LIM motif. Sciellin cDNA probes hybridize to bands of 3.4 and 4.4 kilobase pairs on Northern blots of cultured human keratinocyte RNA. The gene was mapped to human chromosome band 13q22 by fluorescence in situ hybridization. Radiation hybrid mapping demonstrated that sciellin is linked to the sequence tagged site marker WI-457 with a logarithm of the odds score of 7.77. In situ hybridization of human foreskin tissue sections demonstrated that sciellin is expressed in the stratum granulosum. Immunofluorescent staining with a polyclonal rabbit antibody made to a recombinant sciellin protein showed peripheral cytoplasmic localization in the upper cell layers of epidermis and in stratified squamous epithelia such as the oral cavity, esophagus, and vagina. Simple and columnar epithelia, with the exception of the amnion, showed no reaction.

Selective Inhibition of Factor Xa in the Prothrombinase Complex by the Carboxyl-terminal Domain of Antistasin

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.

Conformational Change in an Anti-integrin Antibody: Structure of OPG2 Fab Bound to a β3 Peptide

Antibodies are important tools to explore receptor–ligand interactions. The anti-integrin antibody OPG2 binds in an RGD-related manner to the αIIbβ3integrin as a molecular mimic of fibrinogen. The Fab fragment from OPG2 was cocrystallized with a peptide from the β3subunit of the integrin representing a site that binds RGD. The crystal structure of the complex was determined at 2.2-Å resolution and compared with the unbound Fab. On binding the integrin peptide there were conformational changes in CDR3 of the heavy chain. Also, a significant shift across the intermolecular interface between the CH1-CLdomains was observed so that the angle of rotation relating the two domains was reduced by 15°. This unusual conformational adjustment represents the first example of ligand-induced conformational changes in the carboxyl domains of a Fab fragment.

Crystal structure of a recombinant alphaEC domain from human fibrinogen-420.

The crystal structure of a recombinant alphaEC domain from human fibrinogen-420 has been determined at a resolution of 2.1 A. The protein, which corresponds to the carboxyl domain of the alphaE chain, was expressed in and purified from Pichia pastoris cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the beta- and gamma-chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.

Comparison of Recombinant Human PDE4 Isoforms: Interaction with Substrate and Inhibitors

Four cyclic-nucleotide phosphodiesterase (PDE) genes belonging to the PDE4 family (PDE4A, 4B, 4C and 4D) have been identified. All four isogenes, including several deletions and alterations of the amino, carboxyl and central catalytic domains, were expressed in insect cells. Lysates were characterised for enzyme activity by using the K m for substrate and the EC 50 for activation by the cofactor Mg 2+. The catalytic domain alone appears to be sufficient for the normal enzymatic function of PDE4 proteins. Substrate affinity varied by less than 2-fold between catalytic-domain forms of the PDE4A, 4B and 4D isogenes and the long forms (PDE4A5, PDE4B1 and PDE4D3). The affinity for Mg 2+ varied by less than 4-fold between long and catalytic-domain forms of PDE4A and 4B. The catalytic-domain form of PDE4D, however, had a 12-fold lower affinity for Mg 2+ that was restored by including a portion of the amino-terminal domain, upstream conserved region-2 (UCR2). This result suggests that the Mg 2+-binding site of PDE4D involves the UCR2 region. Inhibition of the PDE4 proteins by synthetic compounds is apparently affected differently by the domains. For PDE4B, the catalytic domain is sufficient for interactions with the inhibitors studied: IBMX, trequinsin, rolipram, TVX 2706, RP 73401 and RS-25344. For PDE4D the catalytic-domain form is less sensitive than the long form to inhibition by RS-25344, rolipram and TVX 2706, by 1463-, 11- and 12-fold, respectively. Addition of UCR2 to the catalytic-domain form of PDE4D restored all the lost sensitivities. The catalytic-domain form of PDE4A showed a reduced inhibitor affinity with RS-25344 and TVX 2706 by 77- and 90-fold, respectively. Both catalytic-domain and long forms of PDE4 isogenes interacted with equal affinity with the non-specific inhibitors IBMX and trequinsin, as well as the very potent PDE4-specific inhibitor RP 73401. Other potent and specific PDE4 inhibitors, such as rolipram, RS-25344 or TVX 2706, appear to utilize non-catalytic domain interactions with PDE4D and 4A to supplement those within the catalytic domains. These observations suggest a different relation between amino and catalytic domains in PDE4D relative to PDE4B. We therefore propose a model to illustrate these isogene-specific PDE4 domain interactions with substrate, inhibitors and the co-factor Mg 2+. The model for PDE4D is also discussed in relation to changes in the activation curve for Mg 2+ and sensitivity to RS-25344 that accompany phosphorylation of the long form by protein kinase A [6].

Amerindian Pyruvate Carboxylase Deficiency Is Associated with Two Distinct Missense Mutations

We characterized the pyruvate carboxylase (PC) gene by PCR amplification, subcloning, and sequencing. The coding region has 19 exons and 18 introns spanning approximately 16 kb of genomic DNA. Screening both the cDNA and the gene of individuals with the simple A form of PC deficiency revealed an 1828G-->A missense mutation in 11 Ojibwa and 2 Cree patients and a 2229G-->T transversion mutation in 2 brothers of Micmac origin. Carrier frequency may be as high as 1/10 in some groupings. The two point mutations are located in a region of homology conserved among yeast, rat, and human PC, in the vicinity of the carboxylation domain of the enzyme. These data provide the first characterization of the human PC gene structure, the identification of common pathogenic mutations, and the demonstration of a founder effect in the Ojibwa and Cree patients.

Effects of deletion mutations in the yeast Ces1 protein on cell growth and morphology and on high copy suppression of mutations in mRNA capping enzyme and translation initiation factor 4A.

The homologous Saccharomyces cerevisiae genes CES1 and CES4 act as high copy suppressors of temperature-sensitive mutations of Ceg1p, the yeast mRNA capping enzyme. Neither CES1 nor CES4 is essential for cell growth. We find that a double deletion mutant (Delta ces1 Delta ces4 ) grows at 25-37 degrees C, but not at 16 degrees C. Delta ces1 Delta ces4 cells display gross defects in cell shape and budding even at permissive temperatures. Functional analysis of CES1 deletion mutants defines a 145 amino acid C-terminal segment of the 915 amino acid Ces1 protein that is necessary and sufficient to complement the Delta ces1 Delta ces 4 cold-sensitive phenotype, to restore normal morphology and to suppress the temparature-sensitive mutant ceg1-25 . A 147 amino acid C-terminal segment of the 942 amino acid Ces4 protein is sufficient to carry out these same functions. Within this carboxyl domain Ces1p and Ces4p are 80% identical to one another. We report isolation of CES1 in a separate screen for high copy suppression of a temperature-sensitive mutation (A79V) of the yeast translation initiation factor Tif1p (eIF-4A). Deletion of the N-terminal 249 amino acids of Ces1p abolished tif1-A79V suppressor function. CES4 on a multicopy plasmid was unable to suppress tif1-A79V . We surmise that whereas the carboxyl domains of Ces1p and Ces4p are functionally redundant in controlling cell morphology and in suppressing ceg1-25 , full-length Ces1p and Ces4p evince distinct genetic interactions that are likely mediated by their N-terminal segments.

Cloning of a novel Y-box homology protein (chkYB-1HP) cDNA lacking the cold-shock domain

We have isolated the complete cDNA clone of a novel 262 amino acid Chicken YB-1 Homology Protein (chkYB-1HP) by screening a chicken embryo cDNA expression library. While the chkYB-1HP is identical over its carboxyl-terminal 78 amino acids with the Y-box protein YB-1, it differs strikingly from all other Y-box transcription factors by lacking the cold-shock domain (CSD). We propose that proteins like chkYB-1HP that lack the CSD, but retain the hydrophilic carboxyl domain could regulate Y-box proteins through the formation of heterodimeric complexes.

The herpes simplex virus virulence factor ICP34.5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in ICP34.5, MyD116, and GADD34.

The herpes simplex virus (HSV) virulence factor ICP34.5, the mouse myeloid differentiation protein MyD116, and the hamster growth arrest and DNA damage protein GADD34 share a 63-amino-acid carboxyl domain which has significant homologies to otherwise divergent proteins. Here we report that both ICP34.5 and its cellular homolog MyD116 complex through the conserved domain with proliferating cell nuclear antigen. In addition, HSV infection induces a novel 70-kDa cellular protein detectable by antisera to both ICP34.5 and GADD34, demonstrating that this novel protein possesses homology with the 63-amino-acid conserved domain.

Elucidation of neurophysin/bioligand interactions from molecular modeling.

This is a review of our recent modeling work aimed at: (i) development and assessment of techniques for reliable refinement of low-resolution protein structures and (ii) using these techniques, at solving specific problems pertinent to neurophysin-bioligand interactions. Neurophysins I and II (NPI and NPII) serve in the neurosecretory granules of the posterior pituitary as carrier proteins for the neurophyseal hormones oxytocin (OT) and vasopressin (VP), respectively, until the latter are released into blood. NPs are homologous two-domain, sulphur rich small proteins (93-95 residues, 7 disulphide bridges per monomer), capable of being aggregated. The C2 symmetrical NPI2 and NPII2 homodimers, and the (NPI/OT)2 and (NPII/VP)2 heterotetramers, all believed to be the smallest functional units, were modeled using low-resolution structure information, i.e. the C alpha-carbon coordinates of the homologous NPII/dipeptide complex as a template. The all-atom representations of the models were obtained using the SYBYL suite of programs (by Tripos, Inc.). Subsequently, they were relaxed, using a constrained simulated annealing (CSA) protocol, and submitted to about 100 ps molecular dynamics (MD) in water, using the AMBER 4.1 force field. The (NPI/OT)2 and (NPII/VP)2 structures, averaged after the last 20 ps of MD, were remarkably similar to those recently reported either for NPII/dipeptide or NPII/oxytocin complex in the solid state (Chen et al., 1991, Proc. Natl. Acad. Sci., U.S.A. 88, 4240-4244; Rose et al., 1996, Nature Struct. Biol. 3, 163-169). The results indicate that the 3(10) helices (terminating the amino domains) and the carboxyl domains are more mobile than the remainder of the NP monomers. The hormones become anchored by residues 1-3 and 6 to the host, leaving residues 4-5 and 7-9 exposed on the surface and free to move. A cluster of attractive interactions, extending from the ligand binding site, Tyr-24-Ile-26 of unit 1(2), to the inter-monomer interface Val-36 of unit 1(2), Cys-79 and Ile-72 of unit 2(1), is clearly seen. We suggest that both these interactions as well as the increased mobility of the 3(10) helix and the carboxyl domain may contribute to the allosteric communication between the ligand and the unit1-unit2 interface.