Avian retroviral long terminal repeats bind CCAAT/enhancer-binding protein.

DNA-protein interactions involving enhancer and promoter sequences within the U3 regions of several avian retroviral long terminal repeats (LTRs) were studied by DNase I footprinting. The rat CCAAT/enhancer-binding protein, C/EBP, bound to all four viral LTRs examined. The Rous sarcoma virus binding site corresponded closely to the 5' limit of the LTR enhancer; nucleotides -225 to -188 were protected as a pair of adjacent binding domains. The Fujinami sarcoma virus LTR bound C/EBP at a single site at nucleotides -213 to -195. C/EBP also bound to the promoter region of the enhancerless Rous-associated virus-0 LTR at nucleotides -77 to -57. The avian myeloblastosis virus LTR bound C/EBP at three sites: nucleotides -262 to -246, -154 to -134, and -55 to -39. We have previously observed binding of C/EBP to an enhancer in the gag gene of avian retroviruses. A heat-treated nuclear extract from chicken liver bound to all of the same retroviral sequences as did C/EBP. Alignment of the avian retroviral binding sequences with the published binding sites for C/EBP in two CCAAT boxes and in the simian virus 40, polyoma, and murine sarcoma virus enhancers suggested TTGNNGCTAATG as a consensus sequence for binding of C/EBP. When two bases of this consensus sequence were altered by site-specific mutagenesis of the Rous sarcoma virus LTR, binding of the heat-stable chicken protein was eliminated.     

The structure and interactions of the proline-rich domain of ASPP2

ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFkappaB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFkappaB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFkappaB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.     

Kinetics of Src homology 3 domain association with the proline-rich domain of dynamins: specificity, occlusion, and the effects of phosphorylation

Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.     

Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.     

The cell-specific elastase I enhancer comprises two domains.

Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.     

Chemical-shift-perturbation mapping of the phosphotransfer and catalytic domain interaction in the histidine autokinase CheA from Thermotoga maritima

Regulating the activity of the histidine autokinase CheA is a central step in bacterial chemotaxis. The CheA autophosphorylation reaction minimally involves two CheA domains, denoted P1 and P4. The kinase domain (P4) binds adenosine triphosphate (ATP) and orients the gamma phosphate for phosphotransfer to a reactive histidine on the phosphoacceptor domain (P1). Three-dimensional triple-resonance experiments allowed sequential assignments of backbone nuclei from P1 and P4 domains as well as the P4 assignments within a larger construct, P3P4, which includes the dimerization domain P3. We have used nuclear magnetic resonance chemical-shift-perturbation mapping to define the interaction of P1 and P3P4 from the hyperthermophile Thermotoga maritima. The observed chemical-shift changes in P1 upon binding suggest that the P1 domain is bound by interactions on the side opposite the histidine that is phosphorylated. The observed shifts in P3P4 upon P1 binding suggest that P1 is bound at a site distinct from the catalytic site on P4. These results argue that the P1 domain is not bound in a mode that leads to productive phosphate transfer from ATP at the catalytic site and imply the presence of multiple binding modes. The binding mode observed may be regulatory or it may reflect the binding mode needed for effective transfer of the histidyl phosphate of P1 to the substrate proteins CheY and CheB. In either case, this work describes the first direct observation of the interaction between P1 and P4 in CheA.     

eIF4G, eIFiso4G, and eIF4B bind the poly(A)-binding protein through overlapping sites within the RNA recognition motif domains

The poly(A)-binding protein (PABP), a protein that contains four conserved RNA recognition motifs (RRM1-4) and a C-terminal domain, is expressed throughout the eukaryotic kingdom and promotes translation through physical and functional interactions with eukaryotic initiation factor (eIF) 4G and eIF4B. Two highly divergent isoforms of eIF4G, known as eIF4G and eIFiso4G, are expressed in plants. As little is known about how PABP can interact with RNA and three distinct translation initiation factors in plants, the RNA binding specificity and organization of the protein interaction domains in wheat PABP was investigated. Wheat PABP differs from animal PABP in that its RRM1 does not bind RNA as an individual domain and that RRM 2, 3, and 4 exhibit different RNA binding specificities to non-poly(A) sequences. The PABP interaction domains for eIF4G and eIFiso4G were distinct despite the functional similarity between the eIF4G proteins. A single interaction domain for eIF4G is present in the RRM1 of PABP, whereas eIFiso4G interacts at two sites, i.e. one within RRM1-2 and the second within RRM3-4. The eIFiso4G binding site in RRM1-2 mapped to a 36-amino acid region encompassing the C-terminal end of RRM1, the linker region, and the N-terminal end of RRM2, whereas the second site in RRM3-4 was more complex. A single interaction domain for eIF4B is present within a 32-amino acid region representing the C-terminal end of RRM1 of PABP that overlaps with the N-proximal eIFiso4G interaction domain. eIF4B and eIFiso4G exhibited competitive binding to PABP, supporting the overlapping nature of their interaction domains. These results support the notion that eIF4G, eIFiso4G, and eIF4B interact with distinct molecules of PABP to increase the stability of the interaction between the termini of an mRNA.     

Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity

ASAP family Arf GAPs induce the hydrolysis of GTP bound to the Ras superfamily protein Arf1, regulate cell adhesion and migration and have been implicated in carcinogenesis. The ASAP proteins have a core catalytic domain of PH, Arf GAP and Ank repeat domains. The PH domain is necessary for both biological and catalytic functions of ASAP1 and has been proposed to be integrally folded with the Arf GAP domain. Protection studies and analytical ultracentrifugation studies previously reported indicated that the domains are, at least partly, folded together. Here, using NMR spectroscopy and biochemical analysis, we have further tested this hypothesis and characterized the interdomain interaction. A comparison of NMR spectra of three recombinant proteins comprised of either the isolated PH domain of ASAP1, the Arf GAP and ankyrin repeat domain or all three domains indicated that the PH domain did interact with the Arf GAP and Ank repeat domains; however, we found a significant amount of dynamic independence between the PH and Arf GAP domains, consistent with the interactions being transient. In contrast, the Arf GAP and Ank repeat domains form a relatively rigid structure. The PH-Arf GAP domain interaction partially occluded the phosphoinositide binding site in the soluble protein, but binding studies indicated the PIP2 binding site was accessible in ASAP1 bound to a lipid bilayer surface. Phosphoinositide binding altered the conformation of the PH domain, but had little effect on the structure of the Arf GAP domain. Mutations in a loop of the PH domain that contacts the Arf GAP domain affected PIP2 binding and the K(m) and k(cat) for converting Arf1 GTP to Arf1 GDP. Based on these results, we generated a homology model of a composite PH/Arf GAP/Ank repeat domain structure. We propose that the PH domain contributes to Arf GAP activity by either binding to or positioning Arf1 GTP that is simultaneously bound to the Arf GAP domain.     

Elucidation of different inhibition mechanism of small chemicals on PtdInsP-binding domains using in silico docking experiments

Phosphatidylinositides, most negatively charged lipids in cellular membranes, regulate diverse effector proteins through the interaction with their lipid binding domains. We have previously reported inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P₃ and Btk PH domain. Here, we report that the inhibitory effects of same sets of chemicals on Grp1 PH domain and epsin1 ENTH domain to elucidate diversity of inhibitory mechanisms upon different lipid binding domains. Among the chemicals, chemical 8 showed best inhibition in vitro assay for Grp1 PH domain and epsin1 ENTH domain, and then the interaction between small chemicals and lipid binding domains was further investigated by in silico docking experiments. As a result, it was concluded that the diverse inhibitory effects on different lipid binding domains were dependent on not only the number of interactions between small chemical and domain, but also additional interaction with positively charged surfaces as the secondary binding sites. This finding will help to develop lipid binding inhibitors as antagonists for lipid–protein interactions, and these inhibitors would be novel therapeutic drug candidates via regulating effector proteins involved in severe human diseases.