Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C₁₄-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C₁₄-CoA and can also bind the far more abundant palmitoyl-CoA (C₁₆-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C_18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C₁₆-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C₁₆-CoA. These results indicate that ACBD6 can locally sequester C₁₆-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C₁₆-CoA.     

Ligand binding to the ACBD6 protein regulates the acyl-CoA transferase reactions in membranes

The binding determinants of the human acyl-CoA binding domain-containing protein (ACBD) 6 and its function in lipid renewal of membranes were investigated. ACBD6 binds acyl-CoAs of a chain length of 6 to 20 carbons. The stoichiometry of the association could not be fitted to a 1-to-1 model. Saturation of ACBD6 by C_16:0-CoA required higher concentration than less abundant acyl-CoAs. In contrast to ACBD1 and ACBD3, ligand binding did not result in the dimerization of ACBD6. The presence of fatty acids affected the binding of C_18:1-CoA to ACBD6, dependent on the length, the degree of unsaturation, and the stereoisomeric conformation of their aliphatic chain. ACBD1 and ACBD6 negatively affected the formation of phosphatidylcholine (PC) and phosphatidylethanolamine in the red blood cell membrane. The acylation rate of lysophosphatidylcholine into PC catalyzed by the red cell lysophosphatidylcholine-acyltransferase 1 protein was limited by the transfer of the acyl-CoA substrate from ACBD6 to the acyltransferase enzyme. These findings provide evidence that the binding properties of ACBD6 are adapted to prevent its constant saturation by the very abundant C_16:0-CoA and protect membrane systems from the detergent nature of free acyl-CoAs by controlling their release to acyl-CoA-utilizing enzymes.     

Structural and dynamic characterization of a freestanding acyl carrier protein involved in the biosynthesis of cyclic lipopeptide antibiotics

Friulimicin is a cyclic lipodecapeptide antibiotic that is produced by Actinoplanes friuliensis. Similar to the related lipopeptide drug daptomycin, the peptide skeleton of friulimicin is synthesized by a large multienzyme nonribosomal peptide synthetase (NRPS) system. The LipD protein plays a major role in the acylation reaction of friulimicin. The attachment of the fatty acid group promotes its antibiotic activity. Phylogenetic analysis reveals that LipD is most closely related to other freestanding acyl carrier proteins (ACPs), for which the genes are located near to NRPS gene clusters. Here, we report that the solution NMR structure of apo‐LipD is very similar to other four‐helix bundle forming ACPs from fatty acid synthase (FAS), polyketide synthase, and NRPS systems. By recording NMR dynamics data, we found that the backbone motions in holo‐LipD are more restricted than in apo‐LipD due to the attachment of phosphopantetheine moiety. This enhanced stability of holo‐LipD was also observed in differential scanning calorimetry experiments. Furthermore, we demonstrate that, unlike several other ACPs, the folding of LipD does not depend on the presence of divalent cations, although the presence of Mg²⁺ or Ca²⁺ can increase the protein stability. We propose that small structural rearrangements in the tertiary structure of holo‐LipD which lead to the enhanced stability are important for the cognate enzyme recognition for the acylation reaction. Our results also highlight the different surface charges of LipD and FAS‐ACP from A. friuliensis that would allow the acyl‐CoA ligase to interact preferentially with the LipD instead of binding to the FAS‐ACP.     

Structural modification of acyl carrier protein by butyryl group

Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes known as the type II fatty acid synthase. Acyl carrier protein (ACP) shuttles the acyl intermediates between individual pathway enzymes. In this study, we determined the solution structures of three different forms of ACP, apo‐ACP, ACP, and butyryl‐ACP under identical experimental conditions. The structural studies revealed that attachment of butyryl acyl intermediate to ACP alters the conformation of ACP. This finding supports the more general notion that the attachment of different acyl intermediates alters the ACP structure to facilitate their recognition and turnover by the appropriate target enzymes.     

On the mechanism of activation of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase) in brain myelin

Protein kinase F_A (an activating factor of ATP·Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase F_A is spontaneously active and trypsin-labile, whereas the other form of kinase F_A is inactive and trypsin-resistant, suggesting a different membrane topography with active F_A exposed on the outer face of the myelin membrane and inactivu F_Q buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase F_A became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase F_A was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase F_A interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant F_A could be converted to the active/trypsin-labile F_A in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase F_A in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase F_A in the brain myelin through the phospholipase C-katalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein Kinase F_A may represent one mode of control modulating the activity of kinase F_A in the central nervous system myelin.     

Binding orientation and specificity of calmodulin to rat olfactory cyclic nucleotide-gated ion channel

Calmodulin (CaM), the primary intracellular Ca²⁺ receptor, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Recognition between CaM and its target sequence in rat olfactory cyclic nucleotide-gated ion channel (OLFp) was investigated by circular dichroism (CD), fluorescence, and NMR spectroscopy. Fluorescence data showed the OLFp tightly bound to CaM with a dissociation constant of 12?nM in a 1:1 stoichiometry. Far-UV CD data showed that approximately 60% of OLFp residues formed α-helical structures when associated with CaM. NMR data showed that most of the ¹⁵N–¹H HSQC cross-peaks of the ¹⁵N-labeled CaM not only shifted but also split into two sets of peaks upon association with the OLFp. Our data indicated that the two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not interexchangeable within the NMR time scale. In light of the palindromic sequence of OLFp (FQRIVRLVGVIRDW) for CaM targeting, we proposed that the helical OLFp with C₂ symmetry may bind to CaM in two orientations. This hypothesis is supported by the observation that only one set of ¹⁵N–¹H HSQC cross-peaks of the ¹⁵N-labeled CaM was detected upon association with OLFp-M13 chimeric peptide (OLFMp), a mutated OLFp lacking the palindromic feature. The binding specificity of OLFMp to CaM was restored when the palindromic feature was destroyed. Binding modes of CaM/OLFp and CaM/OLFMp simulated by molecular docking were in accord with their distinct patterns observed in HSQC spectra. Our studies suggest that the palindromic residues in OLFp are crucial for the orientation-specific recognition by CaM.     

Exploiting the co-evolution of interacting proteins to discover interaction specificity

Protein interactions are fundamental to the functioning of cells, and high throughput experimental and computational strategies are sought to map interactions. Predicting interaction specificity, such as matching members of a ligand family to specific members of a receptor family, is largely an unsolved problem. Here we show that by using evolutionary relationships within such families, it is possible to predict their physical interaction specificities. We introduce the computational method of matrix alignment for finding the optimal alignment between protein family similarity matrices. A second method, 3D embedding, allows visualization of interacting partners via spatial representation of the protein families. These methods essentially align phylogenetic trees of interacting protein families to define specific interaction partners. Prediction accuracy depends strongly on phylogenetic tree complexity, as measured with information theoretic methods. These results, along with simulations of protein evolution, suggest a model for the evolution of interacting protein families in which interaction partners are duplicated in coupled processes. Using these methods, it is possible to successfully find protein interaction specificities, as demonstrated for >18 protein families.     

A GX2GX3G motif facilitates acyl chain sequestration by Saccharomyces cerevisiae acyl carrier protein

Saccharomyces cerevisiae acyl carrier protein (ScACP) is a component of the large fungal fatty acid synthase I (FAS I) complex. ScACP comprises two subdomains: a conserved ACP domain that shares extensive structural homology with other ACPs and a unique structural domain. Unlike the metazoan type I ACP that does not sequester the acyl chain, ScACP can partially sequester the growing acyl chain within its hydrophobic core by a mechanism that remains elusive. Our studies on the acyl-ScACP intermediates disclose a unique ¹⁸⁸GX₂GX₃G¹⁹⁵ sequence in helix II important for ACP function. Complete loss of sequestration was observed upon mutation of the three glycines in this sequence to valine (G188V/G191V/G195V), while G191V and G188V/G191V double mutants displayed a faster rate of acyl chain hydrolysis. Likewise, mutation of Thr216 to Ala altered the size of the hydrophobic cavity, resulting in loss of C₁₂- chain sequestration. Combining NMR studies with insights from the crystal structure, we show that three glycines in helix II and a threonine in helix IV favor conformational change, which in turn generate space for acyl chain sequestration. Furthermore, we identified the primary hydrophobic cavity of ScACP, present between the carboxyl end of helix II and IV. The opening of the cavity lies between the second and third turns of helix II and loop II. Overall, the study highlights a novel role of the GX₂GX₃G motif in regulating acyl chain sequestration, vital for ScACP function.     

Tissue specificity and the human protein interaction network

A protein interaction network describes a set of physical associations that can occur between proteins. However, within any particular cell or tissue only a subset of proteins is expressed and so only a subset of interactions can occur. Integrating interaction and expression data, we analyze here this interplay between protein expression and physical interactions in humans. Proteins only expressed in restricted cell types, like recently evolved proteins, make few physical interactions. Most tissue‐specific proteins do, however, bind to universally expressed proteins, and so can function by recruiting or modifying core cellular processes. Conversely, most ‘housekeeping’ proteins that are expressed in all cells also make highly tissue‐specific protein interactions. These results suggest a model for the evolution of tissue‐specific biology, and show that most, and possibly all, ‘housekeeping’ proteins actually have important tissue‐specific molecular interactions.     

Measuring and analyzing tissue specificity of human genes and protein complexes

Proteins and their interactions are essential for the survival of each human cell. Knowledge of their tissue occurrence is important for understanding biological processes. Therefore, we analyzed microarray and high-throughput RNA-sequencing data to identify tissue-specific and universally expressed genes. Gene expression data were used to investigate the presence of proteins, protein interactions and protein complexes in different tissues. Our comparison shows that the detection of tissue-specific genes and proteins strongly depends on the applied measurement technique. We found that microarrays are less sensitive for low expressed genes than high-throughput sequencing. Functional analyses based on microarray data are thus biased towards high expressed genes. This also means that previous biological findings based on microarrays might have to be re-examined using high-throughput sequencing results.     

Sequence requirement and subtype specificity in the high‐affinity interaction between human frizzled and dishevelled proteins

Members of the Wnt family of lipoglycoproteins initiate signaling by binding to Frizzled (Fz) receptors, and the signal is then relayed by Disheveled (Dvl). The Dvl PDZ domain is known to interact directly with a peptide derived from the KTXXXW motif of Fz7, which is conserved in all known Fz subtypes. We found that an extended region spanning the KTXXXW motif on both its N‐terminal and C‐terminal sides dramatically influences the affinity of peptides derived from Fz7 for Dvl PDZ. An alanine scanning study identified the specific residues external to the KTXXXW motif that are important for high‐affinity binding. In a circular dichroism analysis, mutation of some of these critical residues resulted in peptide conformational changes, suggesting that the secondary structure of the peptides contributes to Fz‐Dvl PDZ binding. Of the 10 known Fz subtypes, peptides derived from only Fz1, Fz2, Fz3, Fz4, and Fz7 directly bound to Dvl PDZ domain in our study. Other Fz subtypes, including some known to be involved in Wnt/β‐catenin signaling (Fz5, Fz9), did not bind to Dvl, suggesting that direct interaction with Dvl PDZ does not determine the subtype‐specific functionality of Fz. Molecular modeling and circular dichroism studies indicated that the Fz peptides that bind to Dvl PDZ domain form specific conformations that are different from those of nonbinding peptides.     

Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein,HisJ

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP‐cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L‐histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L‐His and many related naturally occurring compounds. Our data confirm that L‐His is the preferred ligand, but that 1‐methyl‐L‐His and 3‐methyl‐L‐His can also bind, while the dipeptide carnosine binds weakly and D‐histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L‐Arg, homo‐L‐Arg, and post‐translationally modified methylated Arg‐analogs also bind with reasonable avidity, with the exception of symmetric dimethylated‐L‐Arg. In contrast, L‐Lys and L‐Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein‐based reagentless biosensor.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

Chemical and posttranslational modification of Escherichia coli acyl carrier protein for preparation of dansyl-acyl carrier proteins

Escherichia coli acyl carrier protein (ACP) contains a single tyrosine residue at position 71. The combined o-nitration of apo-ACP Y71 by tetranitromethane and reduction to 3-aminotyrosyl-apo-ACP were performed to introduce a specific site for attachment of a dansyl fluorescent label. Conditions for purification and characterization of dansylaminotyrosyl-apo-ACP are reported. Dansylaminotyrosyl-apo-ACP was enzymatically phosphopantetheinylated and acylated in vitro with an overall approximately 30% yield of purified stearoyl-dansylaminotyrosyl-ACP starting from unmodified apo-ACP. The steady-state kinetic parameters k(cat) = 22 min(-1) and K(M) = 2.7 microM were determined for reaction of stearoyl-dansylaminotyrosyl-ACP with stearoyl-ACP Delta(9)-desaturase. These results show that dansylaminotyrosyl-ACP will function well for studying binding interactions with the Delta(9)-desaturase and suggest similar possibilities for other ACP-dependent enzymes. The efficient in vivo phosphopantetheinylation of E. coli apo-ACP by coexpression with holo-ACP synthase in E. coli BL21(DE3) using fructose as the carbon source is also reported.     

N-terminal N-myristoylation of proteins: refinement of the sequence motif and its taxon-specific differences

N-terminal N-myristoylation is a lipid anchor modification of eukaryotic and viral proteins targeting them to membrane locations, thus changing the cellular function of modified proteins. Protein myristoylation is critical in many pathways; e.g. in signal transduction, apoptosis, or alternative extracellular protein export. The myristoyl-CoA:protein N-myristoyltransferase (NMT) recognizes the sequence motif of appropriate substrate proteins at the N terminus and attaches the lipid moiety to the absolutely required N-terminal glycine residue. Reliable recognition of capacity for N-terminal myristoylation from the substrate protein sequence alone is desirable for proteome-wide function annotation projects but the existing PROSITE motif is not practical, since it produces huge numbers of false positive and even some false negative predictions.As a first step towards a new prediction method, it is necessary to refine the sequence motif coding for N-terminal N-myristoylation. Relying on the in-depth study of the amino acid sequence variability of substrate proteins, on binding site analyses in X-ray structures or 3D homology models for NMTs from various taxa, and on consideration of biochemical data extracted from the scientific literature, we found indications that, at least within a complete substrate protein, the N-terminal 17 protein residues experience different types of variability restrictions. We identified three motif regions: region 1 (positions 1-6) fitting the binding pocket; region 2 (positions 7-10) interacting with the NMT's surface at the mouth of the catalytic cavity; and region 3 (positions 11-17) comprising a hydrophilic linker. Each region was characterized by physical requirements to single sequence positions or groups of positions regarding volume, polarity, backbone flexibility and other typical properties of amino acids (http://mendel.imp.univie.ac.at/myristate/). These specificity differences are confined partly to taxonomic ranges and are proposed for the design of NMT inhibitors in pathogenic fungal and protozoan systems including Aspergillus fumigatus, Leishmania major, Trypanosoma cruzi, Trypanosoma brucei, Giardia intestinalis, Entamoeba histolytica, Pneumocystis carinii, Strongyloides stercoralis and Schistosoma mansoni. An exhaustive search for NMT-homologues led to the discovery of two putative entomopoxviral NMTs.     

Tryptophan phosphorescence study of the internal dynamics of human erythrocyte membrane proteins upon spectrin modification

Room-temperature tryptophan phosphorescence has been used analyze the slow (millisecond) internal dynamics of proteins in isolated native human erythrocyte membranes, after removal of 95% of spectrin, and after thermal denaturation of spectrin or medium acidification to pH 6.0–4.0, as well as the internal dynamics of spectrin extracted from the membrane in solution. The integral membrane proteins prove to differ sharply from spectrin in their structural and dynamic state. The millisecond movements of structural elements in integral proteins are considerably hindered as compared with spectrin. Removal of the bulk of spectrin from membranes leads to amplification of slow fluctuations in the structure of integral proteins. This suggests involvement of spectrin in the control of the structural and dynamic state of the erythrocyte membrane proteins. The acidification of the medium to pH 6.0–4.0 decreases the internal dynamics of native membrane proteins, which is explained by the pH-induced aggregation of spectrin. After thermal denaturation of spectrin, there is no pH-induced increase in the rigidity of the structure of membrane proteins.     

Determination of the acyl chain specificity of the bovine liver phosphatidylcholine transfer protein. Application of pyrene-labeled phosphatidylcholine species

The phosphatidylcholine transfer protein from bovine liver has specific binding sites for the sn-1 and sn-2 acyl chains of the phosphatidylcholine molecule [Berkhout, T.A., Visser, A.J. W.G., & Wirtz, K.W.A. (1984) Biochemistry 23, 1505-1513]. In the present study, we have investigated the properties of these binding sites by determining both binding and transfer of several sets of pyrenylphosphatidylcholine species. These sets consisted of positional isomers in which the length of the pyrene-labeled acyl chain (i.e., 5-13 methylene units) or of the unlabeled saturated acyl chain (i.e., 9-19 methylene units) was varied in either the sn-1 or the sn-2 position. Binding studies showed that there was a considerable discrimination between positional isomers with the higher affinity observed for those lipids that carry the pyrenyl chain in the sn-2 position. In addition, the affinity is markedly dependent on the length of the acyl chains; pyrenyl acyl chains of 9 and 11 methylene units and the palmitoyl chain provided the most efficient binding. The affinity of the transfer protein for the strongest bound pyrene lipid was approximately 2.5 times higher than for an average egg phosphatidylcholine molecule. In general, the transfer studies were in agreement with the binding data. However, with some short-chain derivatives, transfer rates were faster than expected on the basis of the binding data. This emphasizes the importance of kinetic factors (i.e., activation energy) in the transfer process. The rates of spontaneous transfer decreased monotonically with increasing chain length and were very similar for all positional isomer pairs studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural homology of spinach acyl carrier protein and Escherichia coli acyl carrier protein based on NMR data

Acyl carrier proteins (ACPs) from spinach and from Escherichia coli have been used to demonstrate the utility of proton NMR for comparison of homologous structures. The structure of E. coli ACP had been previously determined and modeled as a rapid equilibrium among multiple conformational forms (Kim and Prestegard, Biochemistry 28:8792–8797, 1989). Spinach ACP showed two slowly exchanging forms and could be manipulated into one form for structural study. Here we compare this single form to postulated multiple forms of E. coli ACP using the limited amount of NOE data available for the spinach protein. A number of long-range NOE contacts were present between homologous residues in both spinach and E. coli ACP, suggesting tertiary structural homology. To allow a more definitive structural comparison, a method was developed to use spinach ACP NOE constraints to search for regions of structural divergence from two postulated forms of E. coli ACP. The homologous regions of the two protein sequences were aligned, additional distance constraints were extracted from the E. coli structure, and these were mapped onto the spinach sequence. These distance constraints were combined with experimental NOE constraints and a distance geometry simulated annealing protocol was used to test for compatibility of the constraints. All of the experimental spinach NOE constraints could be successfully combined with the E. coli data, confirming the general hypothesis of structural homology. A better fit was obtained with one form, suggesting a preferential stabilization of that form in the spinach case. Proteins 27:131–143 © 1997 Wiley-Liss, Inc.     

Comparing side chain packing in soluble proteins,protein‐protein interfaces,and transmembrane proteins

We compare side chain prediction and packing of core and non‐core regions of soluble proteins, protein‐protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high‐resolution crystal structures of these 3 protein classes. We show that the solvent‐inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein‐protein interfaces and in the membrane‐exposed regions of transmembrane proteins can be predicted by the hard‐sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent‐inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within ) up to a relative solvent accessibility, , for all 3 protein classes. Our results show that % of the interface regions in protein complexes are “core”, that is, densely packed with side chain conformations that can be accurately predicted using the hard‐sphere model. We propose packing fraction as a metric that can be used to distinguish real protein‐protein interactions from designed, non‐binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins.