Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (K_D) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with K_D of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain–domain motion in EI can contribute as large as ∼−3.2 kcal/mol toward PEP binding.     

Molecular aspects of the interaction between Mason—Pfizer monkey virus matrix protein and artificial phospholipid membrane

The Mason–Pfizer monkey virus is a type D retrovirus, which assembles its immature particles in the cytoplasm prior to their transport to the host cell membrane. The association with the membrane is mediated by the N‐terminally myristoylated matrix protein. To reveal the role of particular residues which are involved in the capsid‐membrane interaction, covalent labelling of arginine, lysine and tyrosine residues of the Mason–Pfizer monkey virus matrix protein bound to artificial liposomes containing 95% of phosphatidylcholine and 5% phosphatidylinositol‐(4,5)‐bisphosphate (PI(4,5)P₂) was performed. The experimental results were interpreted by multiscale molecular dynamics simulations. The application of these two complementary approaches helped us to reveal that matrix protein specifically recognizes the PI(4,5)P₂ molecule by the residues K20, K25, K27, K74, and Y28, while the residues K92 and K93 stabilizes the matrix protein orientation on the membrane by the interaction with another PI(4,5)P₂ molecule. Residues K33, K39, K54, Y66, Y67, and K87 appear to be involved in the matrix protein oligomerization. All arginine residues remained accessible during the interaction with liposomes which indicates that they neither contribute to the interaction with membrane nor are involved in protein oligomerization. Proteins 2016; 84:1717–1727. © 2016 Wiley Periodicals, Inc.     

Correlation between the interaction of the vinculin tail domain with lipid membranes,its phosphorylation and cell mechanical behaviour

Cell adhesion and cell–cell contacts are a pre‐requisite for proper metabolism, protein synthesis and cell survival. Integrins are the transmembrane receptors that link the extracellular matrix via the FAC (focal adhesion complex) with the cytoskeleton. Vinculin is a pivotal FAC protein that has not only been implicated in regulating FAC formation and transmitting mechanical forces, but also in associating with membranous lipids in biological systems.     

Evidence from NMR interaction studies challenges the hypothesis of direct lipid transfer from L‐FABP to malaria sporozoite protein UIS3

UIS3 is a malaria parasite protein essential for liver stage development of Plasmodium species, presumably localized to the membrane of the parasitophorous vacuole formed in infected cells. It has been recently proposed that the soluble domain of UIS3 interacts with the host liver fatty acid binding protein (L‐FABP), providing the parasite with a pathway for importing exogenous lipids required for its rapid growth. This finding may suggest novel strategies for arresting parasite development. In this study, we have investigated the interaction between human L‐FABP and the soluble domain of Plasmodium falciparum UIS3 by NMR spectroscopy. The amino acid residue‐specific analysis of ¹H,¹⁵N‐2D NMR spectra excluded the occurrence of a direct interaction between L‐FABP (in its unbound and oleate‐loaded forms) and Pf‐UIS3. Furthermore, the spectrum of Pf‐UIS3 was unchanged when oleate or phospholipids were added. The present investigation entails a reformulation of the current model of host‐pathogen lipid transfer, possibly redirecting research for early intervention against malaria.     

Target of rapamycin FATC domain as a general membrane anchor: The FKBP‐12 like domain of FKBP38 as a case study

Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water‐soluble and binds to different membrane mimetics would find broad application. The 33‐residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506‐binding region of the protein FKBP38 (FKBP38‐BD) and used ¹H–¹⁵N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C‐terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6–8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl₂ and CaCl₂). The high water‐solubility of y1fatc enables its use for titration experiments against a membrane‐localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C‐terminal 17–11 residues of the 33‐residue long domain by 1D ¹H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to ¹⁵N‐labeled target protein for NMR studies.     

Molecular dynamics simulation study on the interaction of collagen‐like peptides with gelatinase‐A (MMP‐2)

Although several models have been proposed for the interaction of collagen with gelatinase‐A (matrix metalloproteinases‐2 (MMP‐2)), the extensive role of each domain of gelatinase A in hydrolyzing the collagens with and without interruptions is still elusive. Molecular docking, molecular dynamics (MD) simulation, normal mode analysis (NMA) and framework rigidity optimized dynamics algorithm (FRODAN) based analysis were carried out to understand the function of various domains of MMP‐2 upon interaction with collagen like peptides. The results reveal that the collagen binding domain (CBD) binds to the C‐terminal of collagen like peptide with interruption. CBD helps in unwinding the loosely packed interrupted region of triple helical structure to a greater extent. It can be possible to speculate that the role of hemopexin (HPX) domain is to prevent further unwinding of collagen like peptide by binding to the other end of the collagen like peptide. The catalytic (CAT) domain then reorients itself to interact with the part of the unwound region of collagen like peptide for further hydrolysis. In conclusion the CBD of MMP‐2 recognizes the collagen and aids in unwinding the collagen like peptide with interruptions, and the HPX domain of MMP‐2 binds to the other end of the collagen allowing CAT domain to access the cleavage site. This study provides a comprehensive understanding of the structural basis of collagenolysis by MMP‐2. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 779–794, 2014.     

Molecular dynamics simulations on interaction between bacterial proteins: Implication on pathogenic activities

We perform molecular dynamics simulation studies on interaction between bacterial proteins: an outer‐membrane protein STY3179 and a yfdX protein STY3178 of Salmonella Typhi. STY3179 has been found to be involved in bacterial adhesion and invasion. STY3178 is recently biophysically characterized. It is a soluble protein having antibiotic binding and chaperon activity capabilities. These two proteins co‐occur and are from neighboring gene in Salmonella Typhi‐occurrence of homologs of both STY3178 and STY3179 are identified in many Gram‐negative bacteria. We show using homology modeling, docking followed by molecular dynamics simulation that they can form a stable complex. STY3178 belongs to aqueous phase, while the beta barrel portion of STY3179 remains buried in DPPC bilayer with extra‐cellular loops exposed to water. To understand the molecular basis of interaction between STY3178 and STY3179, we compute the conformational thermodynamics which indicate that these two proteins interact through polar and acidic residues belonging to their interfacial region. Conformational thermodynamics results further reveal instability of certain residues in extra‐cellular loops of STY3179 upon complexation with STY3178 which is an indication for binding with host cell protein laminin.     

GGIP: Structure and sequence‐based GPCR–GPCR interaction pair predictor

G Protein‐Coupled Receptors (GPCRs) are important pharmaceutical targets. More than 30% of currently marketed pharmaceutical medicines target GPCRs. Numerous studies have reported that GPCRs function not only as monomers but also as homo‐ or hetero‐dimers or higher‐order molecular complexes. Many GPCRs exert a wide variety of molecular functions by forming specific combinations of GPCR subtypes. In addition, some GPCRs are reportedly associated with diseases. GPCR oligomerization is now recognized as an important event in various biological phenomena, and many researchers are investigating this subject. We have developed a support vector machine (SVM)‐based method to predict interacting pairs for GPCR oligomerization, by integrating the structure and sequence information of GPCRs. The performance of our method was evaluated by the Receiver Operating Characteristic (ROC) curve. The corresponding area under the curve was 0.938. As far as we know, this is the only prediction method for interacting pairs among GPCRs. Our method could accelerate the analyses of these interactions, and contribute to the elucidation of the global structures of the GPCR networks in membranes. Proteins 2016; 84:1224–1233. © 2016 Wiley Periodicals, Inc.     

NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII

Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap‐binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut‐off host–cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot‐and‐mouth disease virus (FMDV) leader proteinase (Lb^pro), human rhinovirus 2 (HRV2) 2A proteinase (2A^pro) and coxsackievirus B4 (CVB4) 2A^pro with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed ¹³C/¹⁵N sequential backbone assignment of human eIF4GII residues 551–745 and examined their binding to murine eIF4E. eIF4GII_551–745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain‐like Lb^pro only forms a stable complex with eIF4GII_551–745 in the presence of eIF4E, with K_D values in the low nanomolar range; Lb^pro contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin‐like 2A^pro from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K_D values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut‐off.     

Improved sensitivity of firefly luminescent intermediate‐based protein interaction assay using Ser 440 mutant with lower adenylation activity

Protein–protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein–protein interaction assay, the firefly luminescent intermediate‐based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half‐reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin‐induced FKBP12–FRB interactions.     

A method for reverse interactome analysis: High‐resolution mapping of interdomain interaction network in Dam1 complex and its specific disorganization based on the interaction domain expression

An experimental methodology that facilitates functional analysis of numerous protein–protein interactions, which have been found in genome‐wide interactome researches, has long been awaited. We propose herein an antagonistic inhibition‐based approach. The antagonizing polypeptide is generated in the course of interaction domain mapping based on yeast 2‐hybrid (Y2H) screening coupled with in vitro convergence of the Y2H‐selected fragments, which is performed in a formatted procedure. Using the coupled methodology, we first performed a high‐resolution mapping of an interdomain interaction network within budding yeast's Dam1 complex. Dam1 complex is a kinetochore protein complex composed of 10 essential subunits including Spc34p and Spc19p. The high‐resolution mapping revealed the overall network structure within the complex for the first time: Dam1 components form into two separated subnetworks on N‐terminal scaffolding domains of Spc34p and Spc19p, and the coiled‐coil interaction in their C‐terminal domains connects the subnetworks. Secondly, we show that the domain fragments converged in the high‐resolution mapping acted as potent inhibitors for the endogenous interactions when episomally overexpressed. The in vivo Dam1 interaction targeting with the fragments conferred a similar phenotype on the host cells; a critical and irreversible damage, which was accompanied with disturbed budding and chromosome mis‐segregation as a result of disorganized spindle. These phenotypes were strongly related to the cellular function of the Dam1 complex. The results and approach we demonstrated herein not only shed light on the Dam1 molecular architecture but also pave the road to reverse‐interactome analysis and discoveries of novel drugs that target disease‐related protein–protein interactions. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

Vacuolar H⁺‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V₁‐ATPase and membrane‐integral V₀ proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V₁V₀ND). We show that V₁V₀ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.     

Interaction of serum amyloid A with human cystatin C—identification of binding sites

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose natural role seems to be participation in many physiologic and pathological processes. Prolonged increased SAA level in a number of chronic inflammatory and neoplastic diseases gives rise to reactive systemic amyloid A amyloidosis, where the N‐terminal 76‐amino acid residue‐long segment of SAA is deposited as amyloid fibrils. Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been described. Here, we report further evidence corroborating this interaction, and the identification of the SAA and hCC binding sites in the SAA–hCC complex, using a combination of selective proteolytic excision and high‐resolution mass spectrometry. The shortest binding site in the SAA sequence was determined as SAA(86–104), whereas the binding site in hCC sequence was identified as hCC(96–102). Binding specificities of both interacting sequences were ascertained by affinity experiments (ELISA) and by registration of mass spectrum of SAA–hCC complex. Copyright © 2012 John Wiley & Sons, Ltd.     

Activity of a novel‐designed antimicrobial peptide and its interaction with lipids

A new antimicrobial peptide l‐RW containing double amphipathic binding sequences was designed, and its biological activities were investigated in the present study. L‐RW showed antibacterial activity against several bacterial strains but low cytotoxicity to mammalian cells and low hemolytic activity to red blood cells, which makes it a potential and promising peptide for further development. Microscale thermophoresis (MST), a new technique, was applied to study the antimicrobial peptide–lipid interaction for the first time, which examined the binding affinities of this new antimicrobial peptide to various lipids, including different phospholipids, mixture lipids and bacterial lipid extracts. The results demonstrated that l‐RW bound preferentially to negatively charged lipids over neutral lipids, which was consistent with the biological activities, revealing the important role of electrostatic interaction in the binding process. L‐RW also showed higher binding affinity for lipid extract from Staphyloccocus aureus compared with Pseudomonas aeruginosa and Escherichia coli, which were in good agreement with the higher antibacterial activity against S. aureus than P. aeruginosa and E. coli, suggesting that the binding affinity is capable to predict the antibacterial activity to some extent. Additionally, the binding of l‐RW to phospholipids was also performed in fetal bovine serum solution by MST, which revealed that the components in biological solution may have interference with the binding event. The results proved that MST is a useful and potent tool in antimicrobial peptide–lipid interaction investigation. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane-mimetics using GB1 fusion proteins and NMR spectroscopy

The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane-mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid- and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Complex formation dynamics of native and mutated pyrin's B30.2 domain with caspase‐1

Pyrin protein is the product of the MEFV gene, mutations in which cause manifestation of familial Mediterranean fever (FMF). Functions of pyrin are not completely clear. The secondary structure of the pyrin is represented with four domains and two motifs. Mutations p.M680I, p.M694V, p.M694I, p.K695R, p.V726A, and p.A744S, which are located in the B30.2 domain of pyrin protein, are responsible for manifestation of the most common and severe forms of FMF. All the domains and the motifs of pyrin, are directly or indirectly, involved in the protein–protein interaction with proteins of apoptosis and regulate the cascade of inflammatory reactions, which is impaired due to pyrin mutations. It is well known, that malfunction of the pyrin‐caspase‐1 complex is the main reason of inflammation during FMF. Complete tertiary structure of pyrin and the effects of mutations in it are experimentally not studied yet. The aim of this study was to identify possible effects of the abovementioned mutations in the B30.2 domain tertiary structure and to determine their potential consequences in formation of the B30.2‐caspase‐1 complex. Using in silico methods, it was found, that these mutations led to structural rearrangements in B30.2 domain tertiary structure, causing shifts of binding sites and altering the interaction energy between B30.2 and caspase‐1.