212 Shifting interfaces: changes in protein–protein and protein–DNA interfaces probed via molecular dynamics

Over the past decade, there has been a growing interest in studying the binding of DNA to the MutSalpha protein complex. This heterodimeric protein complex, the Msh2/Msh6 complex in humans, is the initial complex that binds mismatched DNA and other DNA defects that occur during replication. This complex has also been shown to bind at least some types of damaged DNA, such as the cross-linked adducts due to the chemotherapeutics cisplatin and carboplatin, or the incorporation of the chemotherapeutic, FdU. As a result of this interest, multiple studies have contrasted the interactions of MutSalpha with its normal mismatched substrate and with the interactions of MutsSalpha with the DNA damaged by chemotherapeutic cisplatin. To complement these studies, we examine the interaction between MutSalpha and the DNA damaged by carboplatin via all-atom molecular dynamics simulations. These simulations provide evidence for subtle changes in the protein–DNA and protein–protein interfaces. The interfaces shifts found are broadly similar to those found in binding with adduct from cis-platin, but have distinct differences. These subtle differences may play a role in the way of the different damages and mismatched DNA are signaled by MutSalpha, and suggest a signaling mechanism for DNA damage that chiefly involves shifts in protein–protein interactions as opposed to changes in protein conformation.     

Nuclear protein in the liver in protein–calorie deficiency

1. The effect of protein–calorie deficiency on nuclear proteins was studied in growing and adult rats by using chemical and electrophoretic methods of fractionation. 2. After 7 days on protein-deficient diets, the amount of neutral-soluble proteins increased, and their electrophoretic pattern changed, with the appearance of a new component. 3. The histone fraction of the liver nuclei in rapidly growing rats was lower than that in either deficient or adult animals. 4. The possible role of nuclear proteins in relation to synthesis of the proteins and nucleic acids of the other parts of the cells is discussed.     

Transient protein–protein complexes in base excision repair

Abstract

Transient protein–protein complexes are of great importance for organizing multiple enzymatic reactions into productive reaction pathways. Base excision repair (BER), a process of critical importance for maintaining genome stability against a plethora of DNA-damaging factors, involves several enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases and accessory proteins acting sequentially on the same damaged site in DNA. Rather than being assembled into one stable multisubunit complex, these enzymes pass the repair intermediates between them in a highly coordinated manner. In this review, we discuss the nature and the role of transient complexes arising during BER as deduced from structural and kinetic data. Almost all of the transient complexes are DNA-mediated, although some may also exist in solution and strengthen under specific conditions. The best-studied example, the interactions between DNA glycosylases and AP endonucleases, is discussed in more detail to provide a framework for distinguishing between stable and transient complexes based on the kinetic data.

Communicated by Ramaswamy H. Sarma     

Role for protein–protein interaction databases in human genetics

Proteomics and the study of protein–protein interactions are becoming increasingly important in our effort to understand human diseases on a system-wide level. Thanks to the development and curation of protein-interaction databases, up-to-date information on these interaction networks is accessible and publicly available to the scientific community. As our knowledge of protein–protein interactions increases, it is important to give thought to the different ways that these resources can impact biomedical research. In this article, we highlight the importance of protein–protein interactions in human genetics and genetic epidemiology. Since protein–protein interactions demonstrate one of the strongest functional relationships between genes, combining genomic data with available proteomic data may provide us with a more in-depth understanding of common human diseases. In this review, we will discuss some of the fundamentals of protein interactions, the databases that are publicly available and how information from these databases can be used to facilitate genome-wide genetic studies.     

Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein–protein docking

EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.     

Data mining for protein–protein interactions in invertebrate model organisms

Well-annotated genome databases are available for many invertebrate species, notably the fruitfly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. An adequate interpretation of this information at the biological level requires the exploration of the interactions between the gene products. Knowledge of protein interactions and the components of cell signalling pathways in the fly and worm are particularly valuable as hypotheses can be rapidly tested using the powerful genetic toolkits available. Invertebrates offer additional experimental advantages when attempting to characterise protein–protein interactions (PPIs). Their relatively small genome size compared to mammals helps to reduce missed interactions due to redundancy, and their function can be addressed using forward (mutants) and reverse (RNA interference) genetics. However, the researcher looking for evidence of PPIs for a protein of interest is faced with the challenge of extracting interaction data from sources that are highly varied, such as the results of microarray experiments in the unstructured text of research papers. This challenge is greatly reduced by a range of public databases of curated information, as well as publicly available, enhanced search engines, which can provide either direct experimental evidence for a PPI, or valuable clues for generating new hypotheses.     

Analysis of secondary structural and physicochemical changes in protein–protein complexes

Conformation switching in protein–protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein–protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein–protein modeling and docking studies.     

LPS–protein aggregation influences protein partitioning in aqueous two-phase micellar systems

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS–protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM_LPS?>?98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K _GFPuv?<?1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein–LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein–LPS aggregation.     

Copper–zinc cross-modulation in prion protein binding

In this paper we report a systematic XAS study of a set of samples in which Cu(II) was progressively added to complexes in which Zn(II) was bound to the tetra-octarepeat portion of the prion protein. This work extends previous EPR and XAS analysis in which, in contrast, the effect of adding Zn(II) to Cu(II)–tetra-octarepeat complexes was investigated. Detailed structural analysis of the XAS spectra taken at both the Cu and Zn K-edge when the two metals are present at different relative concentrations revealed that Zn(II) and Cu(II) ions compete for binding to the tetra-octarepeat peptide by cross-regulating their relative binding modes. We show that the specific metal–peptide coordination mode depends not only, as expected, on the relative metal concentrations, but also on whether Zn(II) or Cu(II) was first bound to the peptide. In particular, it seems that the Zn(II) binding mode in the absence of Cu(II) is able to promote the formation of small peptide clusters in which triplets of tetra-octarepeats are bridged by pairs of Zn ions. When Cu(II) is added, it starts competing with Zn(II) for binding, disrupting the existing peptide cluster arrangement, despite the fact that Cu(II) is unable to completely displace Zn(II). These results may have a bearing on our understanding of peptide-aggregation processes and, with the delicate cross-regulation balancing we have revealed, seem to suggest the existence of an interesting, finely tuned interplay among metal ions affecting protein binding, capable of providing a mechanism for regulation of metal concentration in cells.     

ModuleSearch: finding functional modules in a protein–protein interaction network

Many biological processes are performed by a group of proteins rather than by individual proteins. Proteins involved in the same biological process often form a densely connected sub-graph in a protein–protein interaction network. Therefore, finding a dense sub-graph provides useful information to predict the function or protein complex of uncharacterised proteins in the sub-graph. We developed a heuristic algorithm that finds functional modules in a protein–protein interaction network and visualises the modules. The algorithm has been implemented in a platform-independent, standalone program called ModuleSearch. In an interaction network of yeast proteins, ModuleSearch found 366 overlapping modules. Of the modules, 71% have a function shared by more than half the proteins in the module and 58% have a function shared by all proteins in the module. Comparison of ModuleSearch with other programs shows that ModuleSearch finds more sub-graphs than most other programs, yet a higher proportion of the sub-graphs correspond to known functional modules. ModuleSearch and sample data are freely available to academics at http://bclab.inha.ac.kr/ModuleSearch.     

Structural analysis of protein–protein interactions in type I polyketide synthases

Abstract

Polyketide synthases (PKSs) are responsible for synthesizing a myriad of natural products with agricultural, medicinal relevance. The PKSs consist of multiple functional domains of which each can catalyze a specified chemical reaction leading to the synthesis of polyketides. Biochemical studies showed that protein–substrate and protein–protein interactions play crucial roles in these complex regio-/stereo-selective biochemical processes. Recent developments on X-ray crystallography and protein NMR techniques have allowed us to understand the biosynthetic mechanism of these enzymes from their structures. These structural studies have facilitated the elucidation of the sequence–function relationship of PKSs and will ultimately contribute to the prediction of product structure. This review will focus on the current knowledge of type I PKS structures and the protein–protein interactions in this system.     

Evolutionarily conserved motifs and modules in mitochondrial protein–protein interaction networks

Advances in organelle interactomics have led to new insights into organelle functions. In this study, we considered the common mitochondrial PIN of four evolutionarily distant eukaryotic species, namely Homo sapiens, Mus musculus, Drosophila melanogaster and Caenorhabditis elegans. By comparative interactomics analysis of mitochondrial PINs in these organisms, five conserved modules were identified. Modules comprise the main mitochondrial tasks, including proteins involved in translation process, mitochondrial import inner membrane proteins, TCA cycle enzymes, mitochondrial electron transport chain, and metabolic enzymes. Furthermore, we reemphasize that subgraphs of network, i.e., motifs and themes, may represent evolutionarily conserved topological units which are biologically significant.     

Protein–protein interactions of huntingtin in the hippocampus

Molecular Biology - Huntingtin (HTT) occurs in the neuronal cytoplasm and can interact with structural elements of synapses. Huntington’s disease (HD) results from pathological expansion of a...     

Advances in the study of protein–DNA interaction

Protein-DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein-DNA. Recent developments of these methods and research achievements have been reviewed in this paper.     

Advances in protein–amino acid nutrition of poultry

The ideal protein concept has allowed progress in defining requirements as well as the limiting order of amino acids in corn, soybean meal, and a corn–soybean meal mixture for growth of young chicks. Recent evidence suggests that glycine (or serine) is a key limiting amino acid in reduced protein [23% crude protein (CP) reduced to 16% CP] corn–soybean meal diets for broiler chicks. Research with sulfur amino acids has revealed that small excesses of cysteine are growth depressing in chicks fed methionine-deficient diets. Moreover, high ratios of cysteine:methionine impair utilization of the hydroxy analog of methionine, but not of methionine itself. A high level of dietary l-cysteine (2.5% or higher) is lethal for young chicks, but a similar level of dl-methionine, l-cystine or N-acetyl-l-cysteine causes no mortality. A supplemental dietary level of 3.0% l-cysteine (7× requirement) causes acute metabolic acidosis that is characterized by a striking increase in plasma sulfate and decrease in plasma bicarbonate. S-Methylmethionine, an analog of S-adenosylmethionine, has been shown to have choline-sparing activity, but it only spares methionine when diets are deficient in choline and(or) betaine. Creatine, or its precursor guanidinoacetic acid, can spare dietary arginine in chicks.