Structure homology and interaction redundancy for discovering virus–host protein interactions

Virus–host interactomes are instrumental to understand global perturbations of cellular functions induced by infection and discover new therapies. The construction of such interactomes is, however, technically challenging and time consuming. Here we describe an original method for the prediction of high‐confidence interactions between viral and human proteins through a combination of structure and high‐quality interactome data. Validation was performed for the NS1 protein of the influenza virus, which led to the identification of new host factors that control viral replication.     

Retinol-binding protein from human urine and its interaction with retinol and prealbumin

In freshly collected urine from a patient with glomerulotubular proteinuria there were two bands which contained retinol-binding proteins. The cathodal band showed fluorescence in the ultraviolet. After extraction with organic solvents only the anodal non-fluorescent band remained. After addition of an excess retinol only one band remained which by mobility corresponded to the cathodal band.The anodal of the two bands was therefore probably the apo form and the cathodal the holo form of the same retinol-binding protein. Their proportions, determined by densitometric scanning were approximately 4/1 (anodal/cathodal band). More than 85% of the retinol-binding protein in the urine bound to prealbumin-Sephrose. The apo retinol-binding protein from urine had the same electrophoretic mobility on agarose gel el-ctrophoresis and the same pattern on isoelectric focusing as an retinol-binding protein prepared from serum. The carboxy-terminal amino acid sequence of the retinol-binding protein from freshly collected urine that bound to prealbumin-Sepharose, was -Arg-Leu. The amino-terminal sequence was Glu-Arg-Asp-Cys-Arg-Val-Ser-X-Phe-Arg-Val-Lys-Glu-Asn-Phe-Asp-Lys-Ala-Arg-Phe-X-Gly-Thr-Trp-Tyr-. This sequence and the amino acid composition are compatible with the view that the retinol-binding protein in urine is the same as in plasma.     

One‐plasmid tunable coexpression for mycobacterial protein–protein interaction studies

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline‐dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two‐protein Mycobacterium tuberculosis stearoyl‐CoA Δ⁹ desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.     

Receptor–ligand and parasite protein–protein interactions in Plasmodium vivax: Analysing rhoptry neck proteins 2 and 4

Elucidating receptor–ligand and protein–protein interactions represents an attractive alternative for designing effective Plasmodium vivax control methods. This article describes the ability of P. vivax rhoptry neck proteins 2 and 4 (RON2 and RON4) to bind to human reticulocytes. Biochemical and cellular studies have shown that two PvRON2‐ and PvRON4‐derived conserved regions specifically interact with protein receptors on reticulocytes marked by the CD71 surface transferrin receptor. Mapping each protein fragment's binding region led to defining the specific participation of two 20 amino acid‐long regions selectively competing for PvRON2 and PvRON4 binding to reticulocytes. Binary interactions between PvRON2 (ligand) and other parasite proteins, such as PvRON4, PvRON5, and apical membrane antigen 1 (AMA1), were evaluated and characterised by surface plasmon resonance. The results revealed that both PvRON2 cysteine‐rich regions strongly interact with PvAMA1 Domains II and III (equilibrium constants in the nanomolar range) and at a lower extent with the complete PvAMA1 ectodomain and Domains I and II. These results strongly support that these proteins participate in P. vivax's complex invasion process, thus providing new pertinent targets for blocking P. vivax merozoites' specific entry to their target cells.     

Optimization of protein–protein docking for predicting Fc–protein interactions

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc‐binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking‐based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X‐ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc.     

SnTox5–Snn5: a novel Stagonospora nodorum effector–wheat gene interaction and its relationship with the SnToxA–Tsn1 and SnTox3–Snn3–B1 interactions

The Stagonospora nodorum–wheat interaction involves multiple pathogen‐produced necrotrophic effectors that interact directly or indirectly with specific host gene products to induce the disease Stagonospora nodorum blotch (SNB). Here, we used a tetraploid wheat mapping population to identify and characterize a sixth effector–host gene interaction in the wheat–S. nodorum system. Initial characterization of the effector SnTox5 indicated that it is a proteinaceous necrotrophic effector that induces necrosis on host lines harbouring the Snn5 sensitivity gene, which was mapped to the long arm of wheat chromosome 4B. On the basis of ultrafiltration, SnTox5 is probably in the size range 10–30 kDa. Analysis of SNB development in the mapping population indicated that the SnTox5–Snn5 interaction explains 37%–63% of the variation, demonstrating that this interaction plays a significant role in disease development. When the SnTox5–Snn5 and SnToxA–Tsn1 interactions occurred together, the level of SNB was increased significantly. Similar to several other interactions in this system, the SnTox5–Snn5 interaction is light dependent, suggesting that multiple interactions may exploit the same pathways to cause disease.     

Solvent interactions and protein dynamics in spin-labeled T4 lysozyme

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a "hinge" residue, which explains the high degree of flexibility and motional freedom at this site.     

Heme-based sensors: theoretical modeling of heme-ligand–protein interactions

Heme proteins are uniquely adapted to bind the important diatomic molecules O(2), NO and CO. An increasing number of heme proteins are being discovered that sense these molecules and thereby regulate a variety of biochemical responses. The interactions of diatomic molecules with heme, and with the surrounding protein, are therefore of great interest. Recent theoretical modeling, using density functional theory, captures many features of these interactions, as exemplified by the well-characterized heme protein myoglobin. Important details, however, especially the mutual influence of the bound diatomic molecule and the proximal ligand, remain to be clarified.     

Dynamic interactions of c-fos protein in serum-stimulated 3T3 cells

The c-fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v-fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c-fos is a member of a family of structurally related proteins which includes the fos-related antigens (fra). We have studied the dynamic state of the c-fos protein with an antibody prepared by immunizing rabbits with a plasmid-encoded fos fusion protein. In serum-stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS-PAGE as a 60-68-kD group of bands corresponding to c-fos, a doublet at 44-45-kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50-kD band corresponding to a fra. We show that although c-fos protein synthesis is only transiently induced by serum, the c-fos protein persists within the cell after its synthesis has ceased, and it decays with a half-life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune-precipitated complex even at times when c-fos is no longer synthesized. These kinetics indicate that even following shutoff of c-fos protein synthesis, p39 is newly synthesized and can complex with c-fos protein or a fos-related antigen. During this time, c-fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c-fos and/or the fos-related antigens over a period of 4-8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c-fos and fra protein interactions for the function of these proteins.     

The structural analysis of protein–protein interactions by NMR spectroscopy

A comprehensive understanding of protein–protein interactions is an important next step in our quest to understand how the information contained in a genome is put into action. Although a number of experimental techniques can report on the existence of a protein– protein interaction, very few can provide detailed structural information. NMR spectroscopy is one of these, and in recent years several complementary NMR approaches, including residual dipolar couplings and the use of paramagnetic effects, have been developed that can provide insight into the structure of protein–protein complexes. In this article, we review these approaches and comment on their strengths and weaknesses.     

Protein–protein interaction at crystal contacts

Packing contacts are crystal artifacts, yet they make use of the same forces that govern specific recognition in protein-protein complexes and oligomeric proteins. They provide examples of a nonspecific protein-protein interaction which can be compared to biologically relevant ones. We evaluate the number and size of pairwise interfaces in 152 crystal forms where the asymmetric unit contains a monomeric protein. In those crystal forms that have no element of 2-fold symmetry, we find that molecules form 8 to 10 pairwise interfaces. The total area of the surface buried on each molecule is large, up to 4400 Ų. Pairwise interfaces bury 200–1200 Ų, like interfaces generated at random in a computer simulation, and less than interfaces in protease-inhibitor or antigen-antibody complexes, which bury 1500 Ų or more. Thus, specific contacts occurring in such complexes extend over a larger surface than nonspecific ones. In crystal forms with 2-fold symmetry, pairwise interfaces are fewer and larger on average than in the absence of 2-fold symmetry. Some bury 1500–2500 Ų, like interfaces in oligomeric proteins, and create “crystal oligomers” which may have formed in the solution before crystallizing. © 1995 Wiley-Liss, 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.     

Purification of a bovine counterpart to human prealbumin and its interaction with a bovine retinol-binding protein

A bovine counterpart to human prealbumin was purified from bovine serum by thiol-disulfide exchange chromatography on thiol-Sepharose 4B and affinity chromatography on human retinol-binding protein linked to Sepharose 4B. The bovine prealbumin had alpha1-mobility on agarose gel electrophoresis at pH 8.6. It has the same molecular weight as human prealbumin on gel filtration and consisted of subunits with a molecular weight of 12 500. This is compatible with a tetrameric structure for the bovine protein. Antiserum against human prealbumin cross-reacted with bovine prealbumin and vice versa. The bovine prealbumin formed at high ionic strength complexes with another bovine serum protein which were dissociated at low ionic strength. This property was used to isolate a protein from bovine serum, by chromatography on bovine prealbumin linked to Sepharose which cross-reacted with antiserum against human retinol-binding protein; had a molecular weight of 21 000 and alpha 2-mobility on agarose gel electrophoresis. It was concluded that the latter protein was a bovine retinol-binding protein.