Functional changes in scavenger receptor binding conformation are induced by charge mutants spanning the entire collagen domain

Macrophage scavenger receptors are trimeric integral membrane proteins that bind a diverse array of negatively charged ligands. They have been shown to play a role in the pathogenesis of atherosclerosis and in host responses to microbial infections. Earlier mutational studies demonstrated that the distal segment of the collagen domain of the receptor was critically important for high affinity ligand binding activity. In this study, mutations spanning the entire collagen domain were generated and binding was assayed in transfected cells, as well as in assays employing a secreted, receptor fusion protein. Many of the distal, positively charged C-terminal residues in the type II collagen domain of the receptor, previously reported to be essential for binding at 37 degreesC, were found not to be critical for binding at 4 degreesC. Conversely, more proximally charged residues of the collagen receptor that have not been previously mutated were shown to have substantial effects on binding that were also temperature-dependent. These data suggest that scavenger receptor ligand recognition depends on more complex conformational interactions, involving charged residues throughout the entire collagen domain, than was previously recognized.     

Calcium binding induces interaction between the N- and C-terminal domains of yeast calmodulin and modulates its overall conformation

The newly identified hemochromatosis gene, HFE, and a candidate iron transporter gene, Nramp2, have been proposed as key factors responsible for the regulation of intestinal iron absorption. Although the exact functions of these proteins in intestinal iron absorption are unknown, HFE may be required for the down-regulation of iron absorption that occurs with increasing iron status, and Nramp2 may up-regulate iron absorption when iron status is low. Thus, we examined whether the expression of the HFE and Nramp2 genes are regulated by iron status in the human intestinal cell line Caco-2. HFE mRNA and HFE protein were increased and Nramp2 mRNA was decreased by increasing cellular iron status in Caco-2 cells. This iron-mediated modulation of mRNA levels was specific to iron. Moreover, super-induction of HFE mRNA in the presence of cycloheximide suggests that HFE gene expression may be controlled by a short-lived repressor protein. HFE and Nramp2 mRNA levels also changed in opposite directions during cellular differentiation. This reciprocal modification of the HFE and Nramp2 gene expression during both iron treatment and cell differentiation in Caco-2 cells is consistent with an opposing role for these proteins in homeostatic regulation of human intestinal iron absorption.     

The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73-77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.     

Substrate binding induces depolymerization of the C-terminal peptide binding domain of murine GRP78/BiP

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5'-(beta, gamma-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.     

Small proline-rich proteins are cross-bridging proteins in the cornified cell envelopes of stratified squamous epithelia

The cornified cell envelope (CE) is a specialized structure which contributes barrier function to stratified squamous epithelial cells. It is composed of an amalgam of several structural proteins that are rendered insoluble by isopeptide bond crosslinking by transglutaminases. One set of the structural proteins present in CEs of most such epithelia are the small proline rich (SPR) proteins, which are a family of about 12 related structural proteins. We have recovered a large number of peptides containing isopeptide crosslinks, including 236 involving SPR proteins, following proteolysis of CEs isolated from foreskin epidermal tissue and cultured epidermal keratinocytes. Analysis of this database has provided novel information on their function. First, we found that SPRs became crosslinked to many other structural proteins within the CE. Second, multiple glutamine and lysine residues located only on the amino- and carboxy-termini of the SPR proteins were involved in crosslinking, so that the two ends are functionally equivalent. Third, the SPRs functioned as cross-bridging proteins, by directly adjoining other CE structural proteins. In the specialized case of the epidermal CE, the SPRs cross-bridged between loricrin. In cultured keratinocytes which make little loricrin and serve as a model for internal stratified squamous epithelia, the SPRs formed extensive cross-bridges among themselves. Thus SPRs are ubiquitous cross-bridging proteins whose differential expression patterns apparently reflect specific barrier requirements of different epithelia.     

Crystallization of the receptor binding domain of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the production of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF.     

A Moloney murine leukemia virus-based retroviral vector pseudotyped by the insect retroviral gypsy envelope can infect Drosophila cells

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. Previous data suggest that gypsy ENV-like ORF3 mediates viral infectivity. We have produced in the 293GP/LNhsp701ucL.3 human cell line a Moloney murine leukemia virus-based retroviral vector pseudotyped by the gypsy ENV-like protein. We have shown by immunostaining that the gypsy envelope protein is produced in 293GP/LNhsp701ucL.3 cells and that vector particles collected from these cells can infect Drosophila cells. Our results provide direct evidence that the infectious property of gypsy is due to its ORF3 gene product.     

Crystal structure of the ligand binding domain of the human nuclear receptor PPARgamma

The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPARgamma isoform in glucose metabolism is illustrated by the fact that anti-diabetic thiazolidinediones have been shown to be bona fide PPARgamma ligands. Here we report the crystal structure of apo-PPARgamma ligand binding domain (LBD) determined to 2.9-A resolution. Although the structure of apo-PPARgamma-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPARgamma LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPARgamma-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the omega-loop is extended in PPARgamma and contains additional structural elements. Taken together, the apo-PPARgamma-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPARgamma as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.     

Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes

Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.     

A study into the effects of protein binding on nucleotide conformation

In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations.     

A new DNA-binding motif in the Skn-1 binding domain-DNA complex

The role of ifosfamide as first-line chemotherapy treatment of non metastatic Ewing's sarcoma of the extremity is still under discussion. The purpose of this paper is to report the results achieved in a neoadjuvant protocol (REN-3) in which ifosfamide, added to the conventional VACA regimen, was employed since the induction phase. Induction chemotherapy consisted of vincristine, cyclophosphamide, doxorubicin, actinomycin-D and ifosfamide. After local treatment, patients received the drugs used in the induction phase and etoposide. Between November 1991 and November 1994, 61 patients with non metastatic Ewing's sarcoma of the extremity were treated. Forty-nine patients underwent surgery and 73.5% of them had a good histologic response. At a median follow-up of 60 months (range 32-76), 48 patients (79%) remained continuously disease-free. The 5-year event-free and overall survival were 77% and 87%, respectively. These results were significantly better both in terms of histologic response or event-free and overall survival than those obtained in 58 patients with non metastatic Ewing's sarcoma of the extremity treated in a previous protocol (REN-2) in which the same drugs were used, but ifosfamide was employed only in the maintenance phase. The present study suggests the importance of early use of ifosfamide in the treatment of patients with non metastatic Ewing's sarcoma of the extremity.     

Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.     

The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system

The surface proteins (SU) of murine type-C retroviruses have a central hypervariable domain devoid of cysteine and rich in proline. This 41-amino-acid region of Friend ecotropic murine leukemia virus SU was shown to be highly tolerant of insertions and deletions. Viruses in which either the N-terminal 30 amino acids or the C-terminal 22 amino acids of this region were replaced by the 7-amino-acid sequence ASAVAGA were fully infectious. Insertions of this 7-amino-acid sequence at the N terminus, center, and the C terminus of the hypervariable domain had little effect on envelope protein (Env) function, while this insertion at a position 10 amino acids following the N terminus partially destabilized the association between the SU and transmembrane subunits of Env. Large, complex domains (either a 252-amino-acid single-chain antibody binding domain [scFv] or a 96-amino-acid V1/V2 domain of HIV-1 SU containing eight N-linked glycosylation sites and two disulfides) did not interfere with Env function when inserted in the center or C-terminal portions of the hypervariable domain. The scFv domain inserted into the C-terminal region of the hypervariable domain was shown to mediate binding of antigen to viral particles, demonstrating that it folded into the active conformation and was displayed on the surface of the virion. Both positive and negative enrichment of virions expressing the V1/V2 sequence were achieved by using a monoclonal antibody specific for a conformational epitope presented by the inserted sequence. These results indicated that the hypervariable domain of Friend ecotropic SU does not contain any specific sequence or structure that is essential for Env function and demonstrated that insertions into this domain can be used to extend particle display methodologies to complex protein domains that require expression in eukaryotic cells for glycosylation and proper folding.     

A proline-rich motif (PPPY) in the Gag polyprotein of Mason-Pfizer monkey virus plays a maturation-independent role in virion release

Virus assembly represents one of the last steps in the retrovirus life cycle. During this process, Gag polyproteins assemble at specific sites within the cell to form viral capsids and induce membrane extrusion (viral budding) either as assembly progresses (type C virus) or following formation of a complete capsid (type B and type D viruses). Finally, the membrane must undergo a fusion event to pinch off the particle in order to release a complete enveloped virion. Structural elements within the MA region of the Gag polyprotein define the route taken to the plasma membrane and direct the process of virus budding. Results presented here suggest that a distinct region of Gag is necessary for virus release. The pp24 and pp16 proteins of the type D retrovirus Mason-Pfizer monkey virus (M-PMV) are phosphoproteins that are encoded in the gag gene of the virus. The pp16 protein is a C-terminally located cleavage product of pp24 and contains a proline-rich motif (PPPY) that is conserved among the Gag proteins of a wide variety of retroviruses. By performing a functional analysis of this coding region with deletion mutants, we have shown that the pp16 protein is dispensable for capsid assembly but essential for virion release. Moreover, additional experiments indicated that the virus release function of pp16 was abolished by the deletion of only the PPPY motif and could be restored when this motif alone was reinserted into a Gag polyprotein lacking the entire pp16 domain. Single-amino-acid substitutions for any of the residues within this motif confer a similar virion release-defective phenotype. It is unlikely that the function of the proline-rich motif is simply to inhibit premature activation of protease, since the PPPY deletion blocked virion release in the context of a protease-defective provirus. These results demonstrate that in type D retroviruses a PPPY motif plays a key role in a late stage of virus budding that is independent of and occurs prior to virion maturation.