What makes a binding site a binding site?

Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site.     

Diacylglycerol kinase γ is a novel anionic phospholipid binding protein with a selective binding preference

There are ten isozymes of diacylglycerol kinase (DGK), and they regulate diverse patho-physiological functions. Here, we investigated the lipid-binding properties of DGK isozymes using protein–lipid overlay and liposome-binding assays. DGKγ showed a strong binding activity compared with other DGK isozymes for phosphatidic acid (PA) among the various glycerophospholipids tested. However, DGKγ failed to interact with DG and lyso-PA. Moreover, the isozyme was capable of binding to ceramide-1-phosphate but not to ceramide or sphingosine-1-phosphate. The isozyme bound more strongly to PA containing unsaturated fatty acid than to PA having only saturated fatty acid. An analysis using a series of deletion mutants of DGKγ revealed that the N-terminal region, which contains a recoverin homology domain and EF-hand motifs, is responsible for the PA binding activity of DGKγ. Taken together, these results indicate that DGKγ is an anionic phospholipid binding protein that preferably interacts with a small highly charged head group that is very close to the glycerol or sphingosine backbone.     

How does a small molecule bind at a cryptic binding site?

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are “cryptic”: When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands’ binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.     

Presence of a highly efficient “binding” to bacterial contamination can distort data from binding studies

[³H]GABA at low concentrations (5–10 nM) was bound by what appeared to be a GABA receptor binding site in bacterial contamination originating from a batch of distilled water. Under experimental conditions similar to those usually employed in [³H]GABA binding studies, the apparent binding displayed a very high specific component and a high efficiency in terms of [³H]GABA bound per mg of protein. The binding was blocked by muscimol but not by isoguvacine, SR95531 and nipecotic acid. These characteristics suggest that the presence of such spurious binding in the experiments using³H-labeled ligands in brain homogenates may not always be very obvious and, morover, it can result in subtle, but serious, distortions of data from such studies, which may not be immediately recognized.     

VEGF-A121a binding to Neuropilins – A concept revisited

All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue^{1 Kut C, Mac Gabhann F, Popel AS. Where is VEGF in the body? A meta-analysis of VEGF distribution in cancer. Br J Cancer. 2007;97:978-85. doi:10.1038/sj.bjc.6603923. PMID:17912242.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]}, and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs.^{2 Guo HF, Vander Kooi CW. Neuropilin Functions as an Essential Cell Surface Receptor. J Biol Chem. 2015;290:29120-6. doi:10.1074/jbc.R115.687327. PMID:26451046.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]} There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms – that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a – bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not ‘bridge’ VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.     

Do antibody binding techniques identify polysomes synthesizing a specific protein?

Specific binding of antibody directed against MOPC-21 myeloma protein to MOPC-21 polysomes has been demonstrated. It was also shown that this same antibody bound specifically to the ribosomal subunits of these polysomes, suggesting that the antibody is not binding exclusively to the nascent polypeptide chains on the polysomes. The antibody was bound specifically to polysomes of a nonproducing variant XC1 which had been isolated in the presence of MOPC-21 myeloma protein while the antibody was not bound to XC1 polysomes isolated in the absence of myeloma protein. This suggests that the myeloma protein is adsorbed to the polysomes during the isolation procedure.     

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [¹⁵N-¹H]- and [¹³C-¹H]-methyl-TROSY NMR spectra with a 52?kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48?h at a temperature of 25?°C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan ¹⁵N backbone positions and also resolved signals for ¹⁵N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [¹³C-¹H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.     

Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin?

The opportunistic human pathogen Staphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidis attachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureus collagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus and S. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the alpha(1) integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin.     

Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

Peloruside A is a microtubule-stabilizing macrolide that binds to beta tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D, and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10-15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G2/M arrest in the Pel cell lines at concentrations 10-15 times that required in the parental line. The cells show notable changes in morphology compared to the parental line.     

Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

Peloruside A is a microtubule-stabilizing macrolide that binds to β-tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10–15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G₂/M arrest in the Pel cell lines at concentrations 10–15 times that required in the parental line. The cells show notable changes in morphology compared with the parental line.Key words: Apeloruside A, laulimalide, paclitaxel, drug resistance, mitotic arrest, binding site, β-tubulin     

What is an antidepressant binding site doing in a bacterial transporter?

LeuT is a bacterial amino acid transporter belonging to a large family of membrane proteins, including the neurotransmitter transporters that are targets for antidepressant drugs. The high-resolution structure of LeuT has provided an important model for understanding structure and function in this family. Two recent papers found that LeuT can bind tricyclic antidepressants, raising the possibility that it may also serve as a model for the pharmacological properties of neurotransmitter transporters.     

The importin β binding domain as a master regulator of nucleocytoplasmic transport

Specific and efficient recognition of import cargoes is essential to ensure nucleocytoplasmic transport. To this end, the prototypical karyopherin importin β associates with import cargoes directly or, more commonly, through import adaptors, such as importin α and snurportin. Adaptor proteins bind the nuclear localization sequence (NLS) of import cargoes while recruiting importin β via an N-terminal importin β binding (IBB) domain. The use of adaptors greatly expands and amplifies the repertoire of cellular cargoes that importin β can efficiently import into the cell nucleus and allows for fine regulation of nuclear import. Accordingly, the IBB domain is a dedicated NLS, unique to adaptor proteins that functions as a molecular liaison between importin β and import cargoes. This review provides an overview of the molecular role played by the IBB domain in orchestrating nucleocytoplasmic transport. Recent work has determined that the IBB domain has specialized functions at every step of the import and export pathway. Unexpectedly, this stretch of ~ 40 amino acids plays an essential role in regulating processes such as formation of the import complex, docking and translocation through the nuclear pore complex (NPC), release of import cargoes into the cell nucleus and finally recycling of import adaptors and importin β into the cytoplasm. Thus, the IBB domain is a master regulator of nucleocytoplasmic transport, whose complex molecular function is only recently beginning to emerge. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Allosterism in membrane binding: a common motif of the annexins?

Annexins are a family of proteins generally described as Ca(2+)-dependent for phospholipid binding. Yet, annexins have a wide variety of binding behaviors and conformational states, some of which are lipid-dependent and Ca(2+)-independent. We present a model that captures the cation and phospholipid binding behavior of the highly conserved core of the annexins. Experimental data for annexins A4 and A5, which have short N-termini, were globally modeled to gain an understanding of how the lipid-binding affinity of the conserved protein core is modulated. Analysis of the binding behavior was achieved through use of the lanthanide Tb(3+) as a Ca(2+) analogue. Binding isotherms were determined experimentally from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb(3+) in the presence or absence of membranes. In the presence of lipid, the affinity of annexin for cation increases, and the binding isotherms change from hyperbolic to weakly sigmoidal. This behavior was modeled by isotherms derived from microscopic binding partition functions. The change from hyperbolic to sigmoidal binding occurs because of an allosteric transition from the annexin solution state to its membrane-associated state. Protein binding to lipid bilayers renders cation binding by annexins cooperative. The two annexin states denote two affinities of the protein for cation, one in the absence and another in the presence of membrane. In the framework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability of the protein to undergo the postulated two-state transition.     

Is nitrocellulose filter binding really a universal assay for protein-DNA interactions?

The ability to bind to nitrocellulose is commonly accepted as being a universal property of proteins and has been widely used in many different fields of study. This property was first exploited in the study of DNA-binding proteins 30 years ago, in studies involving DNA binding by the lactose repressor (LacR) of Escherichia coli. Termed the filter-binding assay, it remains the quickest and easiest assay available for the study of protein-DNA interactions. However, the exact mechanism by which proteins bind to nitrocellulose remains uncertain. Given the supposedly universal nature of the interaction, we were surprised to notice that certain LacR variants were completely unable to bind simultaneously to DNA containing a single lac operator and nitrocellulose. Investigation of this loss of binding suggests that LacR requires a protein region that is both hydrophobic in nature and more or less unstructured, in order to bind to both nitrocellulose and DNA. In the case of wild-type, tetrameric LacR, the DNA-recognition domain that is not bound to DNA suffices. Dimeric LacR variants will only bind if they have certain C-terminal extensions. These experiments sound a cautionary note for the use of filter binding as an assay of choice, particularly in applications involving screening for the DNA-binding site of putative DNA-binding proteins.     

The BAR-domain family of proteins: a case of bending and binding?

BAR-domains recently took centre stage in science through a report on the crystal structure of this domain in Drosophila Amphiphysin. Though only weakly conserved at the sequence level, the structure of the BAR domain shows striking similarity to the GTPase-binding domain of Arfaptin 2, an effector of Rho- and Arf- GTPases. On the basis of this sequence and structural similarity, these two proteins have been classified as belonging to the same family, the BAR-domain family, and they probably also have similar functional characteristics. Presented here are the results of a database search for the sequence of the BAR domain of Amphiphysin and Arfaptin 2. This search identified a variety of related proteins, most of which are involved in intracellular transport and especially in endocytosis. For example, the BAR-domain family includes Endophilins, GTPase-activating proteins of the Centaurinbeta family and Oligophrenins, the adaptor proteins APPL1 and APPL2 that were recently shown to interact with the small GTPase Rab5, as well as members of the Sorting nexin family. On the basis of the structures of Amphiphysin and Arfaptin 2 and the cellular role of Amphiphysins in the early steps of endocytosis, the functions of the BAR domain have been defined as a dimerization motif and as sensing and inducing membrane curvature. However, data on Arfaptin 2 and now also on the Adaptor proteins APPL1 and 2 suggest that another function of the BAR domain is to bind to small GTPases.     

Is a nitrogen atom an important pharmacophoric element in sigma ligand binding?

A lingering question in sigma receptor ligand development is whether a nitrogen atom serves as an important pharmacophoric element in binding affinity. To address this question, we have synthesized several phenylalkylpiperidines and phenylalkylpiperazines and demonstrated that removal of the N atom from a typical phenylalkylpiperidine led to little or no binding to sigma receptors. In addition, where two N atoms occur in a compound, such as with phenylalkylpiperazines, the N atom on the longer alkyl chain appears to be more important. Thus, based on this study, the N atom is an important pharmacophoric element in the binding of phenylalkylpiperidines and phenylalkylpiperazines to sigma receptors.     

Characteristics of a nickel–albumin binding assay for assessment of myocardial ischaemia

Background: The aim of this study was to describe a method to measure ischaemia-induced alterations of the binding capacity of serum albumin to exogenous nickel.

Methods: We measured the levels of cardiac troponin I (cTnI), serum albumin, ischaemia-modified albumin (IMA) measured by a cobalt–albumin binding assay (CABA), and a nickel–albumin binding assay (NABA) in the following groups: myocardial infarction (n?=?32) and non-ischaemic chest pain (n?=?64).

Results: IMA, cTnI and NABA levels were higher in the myocardial infarction group. NABA presented a higher ability to discriminate myocardial ischaemia than CABA.

Conclusions: Patients with myocardial infarction have reduced nickel binding to human serum albumin, and NABA may have an important role as an early marker of myocardial ischaemia.