Inositol phosphates compete with nucleic acids for binding to bovine leukemia virus matrix protein: Implications for deltaretroviral assembly

The matrix (MA) domain of retroviral Gag proteins plays a crucial role in virion assembly. In human immunodeficiency virus type 1 (HIV‐1), a lentivirus, the presence of phosphatidylinositol‐(4,5)‐bisphosphate triggers a conformational change allowing the MA domain to bind the plasma membrane (PM). In this study, the MA protein from bovine leukemia virus (BLV) was used to investigate the mechanism of viral Gag binding to the membrane during replication of a deltaretrovirus. Fluorescence spectroscopy was used to measure the binding affinity of MA for two RNA constructs derived from the BLV genome as well as for single‐stranded DNA (ssDNA). The importance of electrostatic interactions and the ability of inositol hexakisphosphate (IP6) to compete with nucleic acids for binding to MA were also investigated. Our data show that IP6 effectively competes with RNA and DNA for BLV MA binding, while [NaCl] of greater than 100 mM is required to produce any observable effect on DNA‐MA binding. These results suggest that BLV assembly may be highly dependent on the specific interaction of the MA domain with components of the PM, as observed previously with HIV‐1. The mode of MA binding to nucleic acids and the implications for BLV assembly are discussed. Proteins 2013; 81:1377–1385. © 2013 Wiley Periodicals, Inc.     

Mass spectrometry-based footprinting of protein–protein interactions

We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein–protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein–protein interactions that are less amenable for crystallographic or NMR analysis.     

Quantitative prediction of protein–protein binding affinity with a potential of mean force considering volume correction

Quantitative prediction of protein–protein binding affinity is essential for understanding protein–protein interactions. In this article, an atomic level potential of mean force (PMF) considering volume correction is presented for the prediction of protein–protein binding affinity. The potential is obtained by statistically analyzing X‐ray structures of protein–protein complexes in the Protein Data Bank. This approach circumvents the complicated steps of the volume correction process and is very easy to implement in practice. It can obtain more reasonable pair potential compared with traditional PMF and shows a classic picture of nonbonded atom pair interaction as Lennard‐Jones potential. To evaluate the prediction ability for protein–protein binding affinity, six test sets are examined. Sets 1–5 were used as test set in five published studies, respectively, and set 6 was the union set of sets 1–5, with a total of 86 protein–protein complexes. The correlation coefficient (R) and standard deviation (SD) of fitting predicted affinity to experimental data were calculated to compare the performance of ours with that in literature. Our predictions on sets 1–5 were as good as the best prediction reported in the published studies, and for union set 6, R = 0.76, SD = 2.24 kcal/mol. Furthermore, we found that the volume correction can significantly improve the prediction ability. This approach can also promote the research on docking and protein structure prediction.     

Determination of nucleic acid by [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb3+ porphyrin as the fluorescence spectral probe in bis(2‐ethylhexyl)sulfosuccinate sodium salt micelle system

A new system for the determination of nucleic acid by rare earth metallic porphyrin of [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb³⁺ [T(3‐MO‐4HP)–Tb³⁺] porphyrin as fluorescence spectral probe has been developed in this paper. Nucleic acid can enhance the fluorescence intensity of the T(3‐MO‐4HP)–Tb³⁺ porphyrin in the presence of bis(2‐ethylhexyl)sulfosuccinate sodium salt(AOT) micelle. In pH 8.00 Tris–HCl buffer solution, under optimum conditions, the enhanced fluorescence intensity is in proportion to the concentration of nucleic acids in the range of 0.05–3.00 µg mL^?1 for calf thymus DNA (ct DNA) and 0.03–4.80 µg mL^?1 for fish sperm DNA(fs DNA). Their detection limits are 0.03 and 0.01 µg mL^?1, respectively. In addition, the binding interaction mechanism between T(3‐MO‐4HP)–Tb³⁺ porphyrin and ct DNA is also investigated by resonance scattering and fluorescence spectra. The maximum binding number is calculated by molar ratio method. The new system can be used for the determination of nucleic acid in pig liver, yielding satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Mutating the charged residues in the binding pocket of cellular retinoic acid-binding protein simultaneously reduces its binding affinity to retinoic acid and increases its thermostability.

Three-dimensional modeling of the complex between retinoic acid-binding protein (CRABP) and retinoic acid suggests that binding of the ligand is mediated by interaction between the carboxyl group of retinoic acid and two charged amino acids (Arg-111 and Arg-131) whose side chains project into the barrel of the protein. To assess the contribution of these amino acids to protein-ligand interaction, amino acid substitutions were made by oligonucleotide-directed, site-specific mutagenesis. The wild-type and mutant proteins were expressed in E. coli and subsequently purified. Like wild-type CRABP, the mutant proteins are composed mainly of beta-strands as determined by circular dichroism in the presence and absence of ligand, and thus presumably are folded into the same compact barrel structure as the wild-type protein. Mutants in which Arg-111 and Arg-131 are replaced by glutamine bind retinoic acid with significantly lower affinity than the wild-type protein, arguing that these two residues indeed interact with the ligand. The mutant proteins are more resistant to thermal denaturation than wild-type CRABP in the absence of retinoic acid, but they are not as thermostable as the CRABP-retinoic acid complex. These data suggest a model for CRABP-retinoic acid interaction in which the repulsive forces between the positively-charged arginine residues provide conformational flexibility to the native protein for retinoic acid to enter the binding pocket. Elimination of the positively-charged pair of amino acids produces a protein that is more thermostable than wild-type CRABP but less effective at ligand-binding.     

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G‐protein complex. We have recently established that in Arabidopsis, the regulator of G‐protein signaling (RGS1) protein and a lipid‐hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase‐activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid‐hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys²⁵⁹) is directly involved in RGS1–PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid‐mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal–response output.     

Using collections of structural models to predict changes of binding affinity caused by mutations in protein–protein interactions

Protein–protein interactions (PPIs) in all the molecular aspects that take place both inside and outside cells. However, determining experimentally the structure and affinity of PPIs is expensive and time consuming. Therefore, the development of computational tools, as a complement to experimental methods, is fundamental. Here, we present a computational suite: MODPIN, to model and predict the changes of binding affinity of PPIs. In this approach we use homology modeling to derive the structures of PPIs and score them using state‐of‐the‐art scoring functions. We explore the conformational space of PPIs by generating not a single structural model but a collection of structural models with different conformations based on several templates. We apply the approach to predict the changes in free energy upon mutations and splicing variants of large datasets of PPIs to statistically quantify the quality and accuracy of the predictions. As an example, we use MODPIN to study the effect of mutations in the interaction between colicin endonuclease 9 and colicin endonuclease 2 immune protein from Escherichia coli. Finally, we have compared our results with other state‐of‐art methods.     

The role of PLDα1 in providing specificity to signal‐response coupling by heterotrimeric G‐protein components in Arabidopsis

Heterotrimeric G‐proteins comprised of Gα, Gβ and Gγ subunits are important signal transducers in all eukaryotes. In plants, G‐proteins affect multiple biotic and abiotic stress responses, as well as many developmental processes, even though their repertoire is significantly limited compared with that in metazoan systems. One canonical and three extra‐large Gα, 1 Gβ and 3 Gγ proteins represent the heterotrimeric G‐protein complex in Arabidopsis, and a single regulatory protein, RGS1, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of signaling components and the range of processes they influence is rather intriguing. We now present evidence that the phospholipase Dα1 protein is a key component and modulator of the G‐protein complex in affecting a subset of signaling pathways. We also show that the same G‐protein subunits and their modulators exhibit distinct physiological and genetic interactions depending on specific signaling and developmental pathways. Such developmental plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine tune the plants' responses to constantly changing environments.