Showing papers on "Protein–protein interaction published in 1998"

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions

Abstract: The tetratricopeptide repeat (TPR) is a degenerate 34 amino acid sequence identified in a wide variety of proteins, present in tandem arrays of 3-16 motifs, which form scaffolds to mediate protein-protein interactions and often the assembly of multiprotein complexes. TPR-containing proteins include the anaphase promoting complex (APC) subunits cdc16, cdc23 and cdc27, the NADPH oxidase subunit p67 phox, hsp90-binding immunophilins, transcription factors, the PKR protein kinase inhibitor, and peroxisomal and mitochondrial import proteins. Here, we report the crystal structure of the TPR domain of a protein phosphatase, PP5. Each of the three TPR motifs of this domain consist of a pair of antiparallel alpha-helices of equivalent length. Adjacent TPR motifs are packed together in a parallel arrangement such that a tandem TPR motif structure is composed of a regular series of antiparallel alpha-helices. The uniform angular and spatial arrangement of neighbouring alpha-helices defines a helical structure and creates an amphipathic groove. Multiple-TPR motif proteins would fold into a right-handed super-helical structure with a continuous helical groove suitable for the recognition of target proteins, hence defining a novel mechanism for protein recognition. The spatial arrangement of alpha-helices in the PP5-TPR domain is similar to those within 14-3-3 proteins.

Glycosaminoglycan‐protein interactions: definition of consensus sites in glycosaminoglycan binding proteins

Abstract: Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 A spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection.

Molecular basis of double‐stranded RNA‐protein interactions: structure of a dsRNA‐binding domain complexed with dsRNA

Abstract: Protein interactions with double-stranded RNA (dsRNA) are critical for many cell processes; however, in contrast to protein-dsDNA interactions, surprisingly little is known about the molecular basis of protein-dsRNA interactions. A large and diverse class of proteins that bind dsRNA do so by utilizing an approximately 70 amino acid motif referred to as the dsRNA-binding domain (dsRBD). We have determined a 1.9 A resolution crystal structure of the second dsRBD of Xenopus laevis RNA-binding protein A complexed with dsRNA. The structure shows that the protein spans 16 bp of dsRNA, interacting with two successive minor grooves and across the intervening major groove on one face of a primarily A-form RNA helix. The nature of these interactions explains dsRBD specificity for dsRNA (over ssRNA or dsDNA) and the apparent lack of sequence specificity. Interestingly, the dsRBD fold resembles a portion of the conserved core structure of a family of polynucleotidyl transferases that includes RuvC, MuA transposase, retroviral integrase and RNase H. Structural comparisons of the dsRBD-dsRNA complex and models proposed for polynucleotidyl transferase-nucleic acid complexes suggest that similarities in nucleic acid binding also exist between these families of proteins.

Calmodulin and calmodulin-binding proteins in plants

Abstract: ▪ Abstract Calmodulin is a small Ca2+-binding protein that acts to transduce second messenger signals into a wide array of cellular responses Plant calmodulins share many structural and functional features with their homologs from animals and yeast, but the expression of multiple protein isoforms appears to be a distinctive feature of higher plants Calmodulin acts by binding to short peptide sequences within target proteins, thereby inducing structural changes, which alters their activities in response to changes in intracellular Ca2+ concentration The spectrum of plant calmodulin-binding proteins shares some overlap with that found in animals, but a growing number of calmodulin-regulated proteins in plants appear to be unique Ca2+-binding and enzymatic activation properties of calmodulin are discussed emphasizing the functional linkages between these processes and the diverse pathways that are dependent on Ca2+ signaling

Bcl-2 and bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer

Abstract: It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.

The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.

Abstract: The products of the mammalian Pbx and Drosophila exd genes are able to interact with Hox proteins specifically and to increase their DNA binding affinity and selectivity. In the accompanying paper we show that Pbx proteins exist as stable heterodimers with a novel homeodomain protein, Prep1. Here we show that Prep1-Pbx interaction presents novel structural features: it is independent of DNA binding and of the integrity of their respective homeodomains, and requires sequences in the N-terminal portions of both proteins. The Prep1-Pbx protein-protein interaction is essential for DNA-binding activity. Prep1-Pbx complexes are present in early mouse embryos at a time when Pbx is also interacting with Hox proteins. The use of different interaction surfaces could allow Pbx to interact with Prep1 and Hox proteins simultaneously. Indeed, we observe the formation of a ternary Prep1-Pbx1-HOXB1 complex on a HOXB1-responsive target in vitro. Interaction with Prep1 enhances the ability of the HOXB1-Pbx1 complex to activate transcription in a cooperative fashion from the same target. Our data suggest that Prep1 is an additional component in the transcriptional regulation by Hox proteins.

Pan1p, yeast eps15, functions as a multivalent adaptor that coordinates protein-protein interactions essential for endocytosis.

Abstract: A genetic screen for factors required for endocytosis in the budding yeast Saccharomyces cerevisiae previously identified PAN1. Pan1p is a homologue of the mammalian protein eps15, which has been implicated in endocytosis by virtue of its association with the plasma membrane clathrin adaptor complex AP-2. Pan1p contains two eps15 homology (EH) domains, a protein–protein interaction motif also present in other proteins that function in membrane trafficking. To address the role of Pan1p and EH domains in endocytosis, a yeast two-hybrid screen was performed using the EH domain–containing region of Pan1p. This screen identified yAP180A, one of two yeast homologues of a class of clathrin assembly proteins (AP180) that exhibit in vitro clathrin cage assembly activity. In vitro binding studies using GST fusion proteins and yeast extracts defined distinct binding sites on yAP180A for Pan1p and clathrin. yAP180 proteins and Pan1p, like actin, localize to peripheral patches along the plasma membrane. Mammalian synaptojanin, a phosphatidylinositol polyphosphate-5-phosphatase, also has been implicated in endocytosis recently, and three synaptojanin-like genes have been identified in yeast. We observed genetic interactions between the yeast SJL1 gene and PAN1, which suggest a role for phosphoinositide metabolites in Pan1p function. Together with other studies, these findings suggest that Pan1p coordinates regulatory interactions between proteins required for both endocytosis and actin-cytoskeleton organization; these proteins include the yAP180 proteins, clathrin, the ubiquitin–protein ligase Rsp5p, End3p, and synaptojanin. We suggest that Pan1p (and by extension eps15) serves as a multivalent adaptor around which dynamic interactions between structural and regulatory components of the endocytic pathway converge.

Differential interactions of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants

Abstract: Hsp90 is required for the normal function of steroid receptors, but its binding to steroid receptors is mediated by Hsc70 and several hsp-associated accessory proteins. An assortment of Hsp90 mutants were tested for their abilities to interact with each of the following accessories: Hop, Cyp40, FKBP52, FKBP51, and p23. Of the 11 Hsp90 mutants tested, all were defective to some extent in associating with progestin (PR) complexes. In every case, however, reduced PR binding correlated with a defect in binding of one or more accessories. Co-precipitation of mutant Hsp90 forms with individual accessories was used to map Hsp90 sequences required for accessory protein interactions. Mutation of Hsp90's highly conserved C-terminal EEVD to AAVD resulted in diminished interactions with several accessory proteins, most particularly with Hop. Deletion of amino acids 661-677 resulted in loss of Hsp90 dimerization and also caused diminished interactions with all accessory proteins. Binding of p23 mapped most strongly to the N-terminal ATP-binding domain of Hsp90 while binding of TPR proteins mapped to the C-terminal half of Hsp90. These results and others further suggest that the N- and C-terminal regions of Hsp90 maintain important conformational links through intramolecular interactions and/or intermolecular influences in homodimers.

Electrospray‐ionization mass spectrometry of intact intrinsic membrane proteins

Abstract: Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.

Yeast two-hybrid: so many interactions, (in) so little time...

Abstract: Protein-protein interactions are essential to cellular mechanisms at all levels in biologically responsive systems. These interactions occur extracellularly and include ligand-receptor interactions, cell adhesion, antigen recognition, and virus-host recognition. Intracellular protein-protein interactions occur in the formation of multi-protein complexes, during the assembly of cytoskeletal elements, and between receptor-effector, as well as effector-effector, molecules of signal transduction pathways. Finally, assembly of transcriptional machinery involves protein interactions. The yeast two-hybrid method is a powerful technique for analyzing these protein-protein interactions. Since the publication of this technique in the late 1980s, the robust nature and far-reaching utility of yeast two-hybrid systems for functional expression library cloning has led to the identification of many novel proteins in all areas of biological life science research. Additionally, two-hybrid techniques provide a rapid and versatile system for the further characterization of discrete protein-protein interactions. Recent variations on the basic system have enabled application well beyond protein pairs, to investigate multi-protein complexes and protein-nucleotide interactions. Yeast two-hybrid methods necessitate expression and subsequent interaction between a "protein of interest" functional pair within the yeast cell, ultimately driving reporter gene expression and thus effectively linking protein-protein interaction(s) to a change in yeast cell phenotype. Functional protein-protein interactions using the two-hybrid techniques have been demonstrated for all levels of cellular biology; however, until recently, extracellular protein-protein interactions were excluded from investigations using this technique. Investigations from several labs have now demonstrated that extracellular proteins can be studied using two-hybrid methods, thereby enabling intense study of extracellular protein partners using the robust nature and the genetic power of yeast.

CD2 and the nature of protein interactions mediating cell‐cell recognition

Abstract: Rapid progress has recently been made in characterising the structures of leukocyte cell-surface molecules. Detailed analyses of the structure and interactions of CD2 were the first involving a molecule that has not been directly linked to antigen recognition in the manner of antigen receptors or co-receptors. It seems highly likely that the properties of ligand binding by CD2 are relevant to the general mechanisms of cell-cell recognition. As an example of biological recognition, the defining characteristic of cell-cell contact is that it involves the simultaneous interaction of hundreds, if not thousands, of molecules. Affinity and kinetic analyses of ligand binding by CD2 indicated that the protein interactions mediating cell-cell contact, whilst highly specific, are much weaker than initially anticipated, probably due to the requirement that such contacts be easily reversible. Simultaneously, in addressing the mechanism of this mode of recognition, structural and mutational studies focussed on the role of charged residues clustered in the ligand-binding face of CD2, yielding the concept that electrostatic complementarity, rather than surface-shape complementarity, is the dominant feature of specific, low-affinity protein recognition at the cell surface by CD2. The crystallographic analysis of the CD2-binding domain of CD58 strongly supports this concept.

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site.

Abstract: The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifi- cations, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architec- ture is distinct from that of any known DNA- or protein- binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly f lexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphor- ylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site. Transcription factor families are defined by highly conserved DNA-binding domains that display similar DNA recognition properties. The means by which individual family members control different genes therefore must be determined by regulatory mechanisms that enhance the specificity of DNA binding. In the ets gene family, which includes at least 18 members in the human genome, partnerships with additional transcription factors, as well as posttranslational modifications, help dictate specificity for distinct targets (1). These regulatory mechanisms converge on a highly conserved '80-aa region termed the Pointed (PNT) domain (2). The PNT domain occurs in approximately one-third of the ets proteins, including Ets-1, Ets-2, GABPa, and TEL from vertebrates, and PNT-P2 and Yan from Drosophila (Fig. 1). This domain is proposed to mediate protein-protein interac- tions and to be regulated by ras-dependent signaling because of the presence of an adjacent mitogen-activated protein (MAP) kinase phosphorylation site (1). In particular, the PNT domain is implicated in the self-association of chimeric onco- proteins, identified in human leukemias, that result from chromosomal translocations of the gene encoding the ets protein TEL with segments of genes encoding several tyrosine kinases or the acute myeloid leukemia (AML)-1B transcrip- tion factor (3-9). To date sequence conservation has defined the PNT do- main, yet it has not been established that this region is a structural module that acts in a biological context. To create a framework for understanding the role of the PNT domain in the regulation of a variety of ets proteins and in the oncogenic potential of TEL fusion proteins, we have characterized struc- turally a fragment of Ets-1 that includes this domain and the adjacent MAP kinase phosphorylation site.

Interaction of Presenilins with the Filamin Family of Actin-Binding Proteins

Abstract: Mutations in presenilin genes PS1 and PS2 account for ∼50% of early-onset familial Alzheimer’s disease (FAD). The PS1 and PS2 genes encode highly homologous transmembrane proteins related to theCaenorhabditis elegans sel-12 and spe-4 gene products. A hydrophilic loop region facing the cytoplasmic compartment is likely to be functionally important because at least 14 mutations in FAD patients have been identified in this region. We report here that the loop regions of PS1 and PS2 interact with nonmuscle filamin (actin-binding protein 280, ABP280) and a structurally related protein (filamin homolog 1, Fh1). Overexpression of PS1 appears to modify the distribution of ABP280 and Fh1 proteins in cultured cells. A monoclonal antibody recognizing ABP280 and Fh1 binds to blood vessels, astrocytes, neurofibrillary tangles, neuropil threads, and dystrophic neurites in the AD brain. Detection of ABP280/Fh1 proteins in these structures suggests that these presenilin-interacting proteins may be involved in the development of AD and that interactions between presenilins and ABP280/Fh1 may be functionally significant. The ABP280 gene is located on the human X chromosome, whereas the newly identified Fh1 gene maps to human chromosome 3. These results provide a new basis for understanding the function of presenilin proteins and further implicate cytoskeletal elements in AD pathogenesis.

Prediction of functional regions of the maize streak virus replication-associated proteins by protein-protein interaction analysis

Abstract: The replication of the geminiviruses depends on the viral encoded early (complementary-sense) proteins and on host genome encoded factors. Additionally, some of the early proteins (the AL2 protein of subgroup III, and the RepA (formerly known as C1) or Rep (C1:C2) proteins of subgroup I geminiviruses) can function as transcriptional activators of virion- (V-)sense gene expression. The yeast two-hybrid system has allowed us to predict some of the functionally important regions of the maize streak virus (MSV) early proteins RepA and Rep. Defined domains of these proteins were shown to act as transactivators in yeast cells. We detected the association of the RepA and Rep proteins, and their subfragments, with the maize retinoblastoma protein (ZmRb1) which is likely to be one of the interacting host proteins. We showed the self-association capability of the MSV proteins and suggest that homo- or hetero-oligomerization may play an important role in virus replication. These results provide new insights into the role of different regions of the MSV proteins in relation to transcriptional activation and regulation of viral DNA replication.

Maize Endosperm ADP-Glucose Pyrophosphorylase SHRUNKEN2 and BRITTLE2 Subunit Interactions

Abstract: ADP–glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 ( Sh2 ) and Brittle2 ( Bt2 ) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants ( sim ) also identified motifs essential for protein interactions.

Dual recognition and the role of specificity-determining residues in colicin E9 DNase-immunity protein interactions.

Abstract: The immunity protein Im2 can bind and inhibit the noncognate endonuclease domain of the bacterial toxin colicin E9 with a Kd of 19 nM, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 with which it shares 68% sequence identity. Previous work from our laboratory has shown that the specificity differences of these four-helix immunity proteins is due almost entirely to helix II which is largely variable in sequence in the immunity protein family. From alanine scanning mutagenesis of Im9 in conjunction with high-field NMR data, a dual recognition model for colicin−immunity protein specificity has been proposed whereby the conserved residues of helix III of the immunity protein act as the anchor of the endonuclease binding site while the variable residues of helix II control the specificity of the protein−protein interaction. In this work, we identify three residues (at positions 33, 34, and 38) in helix II which define the specificity differences of Im2 and Im9 for colicin E9 and, us...

Interactions within the yeast Sm core complex: from proteins to amino acids.

Abstract: Sm core proteins play an essential role in the formation of small nuclear ribonucleoprotein particles (snRNPs) by binding to small nuclear RNAs and participating in a network of protein interactions. The two-hybrid system was used to identify SmE interacting proteins and to test for interactions between all pairwise combinations of yeast Sm proteins. We observed interactions between SmB and SmD3, SmE and SmF, and SmE and SmG. For these interactions, a direct biochemical assay confirmed the validity of the results obtained in vivo. To map the protein-protein interaction surface of Sm proteins, we generated a library of SmE mutants and investigated their ability to interact with SmF and/or SmG proteins in the two-hybrid system. Several classes of mutants were observed: some mutants are unable to interact with either SmF or SmG proteins, some interact with SmG but not with SmF, while others interact moderately with SmF but not with SmG. Our mutational analysis of yeast SmE protein shows that conserved hydrophobic residues are essential for interactions with SmF and SmG as well as for viability. Surprisingly, we observed that other evolutionarily conserved positions are tolerant to mutations, with substitutions affecting binding to SmF and SmG only mildly and conferring a wild-type growth phenotype.

Activities of the Sex-lethal protein in RNA binding and protein:Protein interactions

Abstract: The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.

NMR for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges.

Abstract: Using the design of bivalent and bridge-binding inhibitors of thrombin as an example, we review an NMR-based experimental approach for the design of functional mimetics of protein-protein interactions. The strategy includes: (i) identification of binding residues in peptide ligands by differential resonance perturbation, (ii) determination of protein-bound structures of peptide ligands by use of transferred NOEs, (iii) minimization of larger protein and peptide ligands on the basis of NMR structural information, and (iv) linkage of two weakly binding mimetics to produce an inhibitor with enhanced affinity and specificity. This approach can be especially effective for the design of potent and selective functional mimetics of protein-protein interactions because it is less likely that the surfaces of two related proteins or enzymes share two identical binding sites or regions.

A Photochemical Switch for Controlling Protein-Protein Interactions.

Abstract: Incorporation of an unnatural amino acid containing a photolabile group in the side chain allows specific interactions between two proteins to be prevented. The photocaged ras protein in which Asp 38 has been substituted by its β-nitrobenzyl ester (Nb) is unable to interact with its effector protein p120-GAP (see drawing below) although it has the same intrinsic GTPase activity. After photocleavage of the Nb group, 50% of the p120-GAP-dependent GTPase activity relative to the wild-type protein is restored.