Quantitatively Characterizing the Ligand Binding Mechanisms of Choline Binding Protein Using Markov State Model Analysis

Protein-ligand recognition plays key roles in many biological processes. One of the most fascinating questions about protein-ligand recognition is to understand its underlying mechanism, which often results from a combination of induced fit and conformational selection. In this study, we have developed a three-pronged approach of Markov State Models, Molecular Dynamics simulations, and flux analysis to determine the contribution of each model. Using this approach, we have quantified the recognition mechanism of the choline binding protein (ChoX) to be ∼90% conformational selection dominant under experimental conditions. This is achieved by recovering all the necessary parameters for the flux analysis in combination with available experimental data. Our results also suggest that ChoX has several metastable conformational states, of which an apo-closed state is dominant, consistent with previous experimental findings. Our methodology holds great potential to be widely applied to understand recognition mechanisms underlining many fundamental biological processes.     

Continuous Evaluation of Ligand Protein Predictions: A Weekly Community Challenge for Drug Docking

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Ligand Binding Reveals a Role for Heme in Translationally-Controlled Tumor Protein Dimerization

The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His⁷⁶His⁷⁷) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP''s cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca²⁺ ligand to TCTP does not alter its monomeric state; although, Ca²⁺ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP''s proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTP''s role as a component of signal transduction mechanisms.     

Mechanism for Attenuation of DNA Binding by MarR Family Transcriptional Regulators by Small Molecule Ligands

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.     

Strong,Specific, Reversible Binding Ligands for Transfer Rna: Comparison By Fluorescence and Nmr Spectroscopies with Distamycin Binding for a New Structural Class of Ligand

Abstract

Binding data are presented for the interaction with brewer's yeast tRNA^Phc of a new structural family of ligands, symmetrical bis-benzimidazoles. In addition specific perturbations in chemical shifts were detected by 1-dimensional NMR spectroscopy at 400 MHz for some imino and aromatic methyl protons of tRNA^Phc when the tRNA was titrated with distamycin. Competitive displacement of the benzimidazole by added distamycin was followed fluorescence spectroscopy.     

Proteomics Approaches for Identification of Tumor Relevant Protein Targets in Pulmonary Squamous Cell Carcinoma by 2D-DIGE-MS

Potential markers for progression of pulmonary squamous cell carcinoma (SCC) were identified by examining samples of lung SCC and adjacent normal tissues using a combination of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF). The PANTHER System was used for gel image based quantification and statistical analysis. An analysis of proteomic data revealed that 323 protein spots showed significantly different levels of expression (P≤0.05) in lung SCC tissue compared to expression in normal lung tissue. A further analysis of these protein spots by MALDI-TOF-MS identified 81 different proteins. A systems biology approach was used to map these proteins to major pathways involved in numerous cellular processes, including localization, transport, cellular component organization, apoptosis, and reproduction. Additionally, the expression of several proteins in lung SCC and normal tissues was examined using immunohistochemistry and western blot. The functions of individual proteins are being further investigated and validated, and the results might provide new insights into the mechanism of lung SCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.     

Three-Dimensional Analysis of CD6 Site-Directed Mutagenesis and Monoclonal Antibody Binding Studies Using the X-ray Structure of Mac-2 Binding Protein and a Molecular Model of the CD6 Ligand Binding Domain

The extracellular region of CD6 consists of three scavenger receptor cysteine-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues important for the CD6-ALCAM interaction have previously been identified by mutagenesis. A total of 22 CD6 residues were classified according to their importance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The three-dimensional structure of the SRCR domain of Mac-2 binding protein has recently been determined, providing a structural prototype for the SRCR protein superfamily. This has made a thorough three-dimensional analysis of CD6 mutagenesis and mAb binding experiments possible. Mutation of buried residues compromised both mAb and ALCAM binding, consistent with the presence of structural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were found to map to the surface in the same region of the domain. This suggests that the CD6 ligand binding site and epitopes of tested mAbs overlap and provides an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the ALCAM binding site.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050263Abbreviations ALCAM activated leukocyte cell adhesion molecule - CD6D3 third (membrane-proxi-mal) extracellular domain of CD6 - IgSF immunoglobulin superfamily - mAb monoclonal antibody - M2BP Mac-2 binding protein - SRCR scavenger receptor cysteine-rich domain - SRCRSF scavenger receptor cysteine-rich protein superfamily     

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T. maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of β(1→4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.     

A Screening Strategy Based on Differential Binding of Ligand to Receptor and to Binding Proteins: Screening for Compounds Interacting with Corticotrophin-Releasing Factor-Binding Protein

The ligand-receptor interaction has been commonly used in development of high throughput screening assays for new drugs. In some cases, an endogenous ligand interacts not only with membrane receptors but also with soluble binding proteins. Corticotrophin-releasing factor (CRF) is an important stress neurotransmitter/hormone involved in both the central and peripheral nervous systems. CRF exerts its function by interacting with CRFR1 and CRFR2 receptors. In addition, CRF-binding protein (CRF-BP) binds CRF with high affinity. Accordingly, CRF-BP has been suggested to play an important role in modulating CRF function. Based on the potential involvement of CRF-BP in many neurological disorders, it is desirable to develop a screening assay to look for drugs that either mimic or interfere with CRF binding to CRF-BP. An assay was developed to monitor the interactions of radiolabeled CRF with human/rat CRF-BP and the mouse CRFR1 (mCRFR1) receptor. By carefully examining the binding characteristics of radiolabeled CRF to mCRFR1, the assay was able to identify compounds that bind to CRF-BP with high affinity and have little or no affinity for mCRFR1 receptors. Based on a mathematical model, we have verified the screening system with several well-characterized CRF ligands that all have different affinities for CRF receptors and CRF-BP.     

Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH₂), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH₂ was uncovered by examining Env expression and maturation in treated cells. GPG-NH₂ treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH₂ negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH₂, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.The endoplasmic reticulum (ER) contains a number of molecular chaperones and folding factors that aid in the maturation of proteins that traverse the secretory pathway. This process is strictly monitored by the ER quality control system, which selects properly folded proteins for export to the Golgi (16) and targets misfolded proteins for destruction through the ER-associated protein degradation pathway (ERAD) (4, 28). Once an ER protein is selected as a substrate for ERAD, it is translocated from the ER lumen to the cytosol through an ER translocon. This retrotranslocation process is thought to be driven by either the cytosolic AAA-ATPase p97 (39) or the 19S proteasome cap (23). Upon entrance into the cytosol, the ERAD substrate is ubquitinated, and its glycans are removed by an N-glycanase to prepare it for proteasomal degradation (11, 28).Viral envelope glycoproteins utilize the host cell secretory pathway for their proper maturation and trafficking to the site of viral assembly. The human immunodeficiency virus type 1 (HIV-1) encodes the envelope glycoprotein (Env), which initiates HIV-1 infections by mediating attachment and fusion of the viral envelope with the host cell membrane (17). Therefore, infectious HIV-1 particle production relies on the ability of Env to pass the rigorous ER quality control system.Env is initially synthesized as a type I membrane precursor glycoprotein termed gp160, which is cotranslationally targeted to the ER by its 30-amino-acid N-terminal signal sequence (24). Within the ER, gp160 receives ∼30 N-linked glycans and is assisted in its maturation by the chaperones BiP, calnexin, and calreticulin as it undergoes extensive disulfide bond formations (15, 21, 31). Once gp160 has reached its native state with ten disulfide bonds and its signal sequence has been cleaved posttranslationally (21, 25), it assembles into trimers (26) and is exported to the Golgi. Within the Golgi, gp160 is cleaved by cellular endoproteases, yielding the transmembrane protein gp41 and the noncovalently associated surface protein gp120 (27). Thereafter, this complex is transported to the plasma membrane, where it is incorporated into the envelope of assembling HIV-1 particles.We have previously shown that a tripeptide amide corresponding to a conserved motif of the HIV-1 Env, glycyl-prolyl-glycine amide (GPG-NH₂), suppressed the replication of all 47 HIV-1 laboratory strains and clinical isolates examined with a 50% inhibitory concentration of ∼10 μM, a concentration that is 200- to 2,000-fold less than what affected cell growth or had other toxic effects on peripheral blood mononuclear cells (35). However, this suppression was not, as we had anticipated, due to interactions of the peptide with the early events of the HIV-1 replication cycle, such as attachment or entry (36). In the present study, we demonstrate that GPG-NH₂ reduced Env incorporation into HIV-1 particles during replication by targeting Env toward the ERAD pathway. The ability of GPG-NH₂ to target Env for degradation was dependent on the presence of functional proteasomes and required the full-length Env signal sequence. These findings illustrate that small molecules may be utilized therapeutically to specifically target unwanted pathogenic proteins for degradation by the existing cellular machinery.     

Crystal Structure of the Pleckstrin Homology Domain from the Ceramide Transfer Protein: Implications for Conformational Change upon Ligand Binding

Ceramide transfer protein (CERT) is responsible for the nonvesicular trafficking of ceramide from the endoplasmic reticulum (ER) to the trans Golgi network where it is converted to sphingomyelin (SM). The N-terminal pleckstrin homology (PH) domain is required for Golgi targeting of CERT by recognizing the phosphatidylinositol 4-phosphate (PtdIns(4)P) enriched in the Golgi membrane. We report a crystal structure of the CERT PH domain. This structure contains a sulfate that is hydrogen bonded with residues in the canonical ligand-binding pocket of PH domains. Our nuclear magnetic resonance (NMR) chemical shift perturbation (CSP) analyses show sulfate association with CERT PH protein resembles that of PtdIns(4)P, suggesting that the sulfate bound structure likely mimics the holo form of CERT PH protein. Comparison of the sulfate bound structure with the apo form solution structure shows structural rearrangements likely occur upon ligand binding, suggesting conformational flexibility in the ligand-binding pocket. This structural flexibility likely explains CERT PH domain’s low affinity for PtdIns(4)P, a property that is distinct from many other PH domains that bind to their phosphoinositide ligands tightly. This unique structural feature of CERT PH domain is probably tailored towards the transfer activity of CERT protein where it needs to shuttle between ER and Golgi and therefore requires short resident time on ER and Golgi membranes.     

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.     

Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP) Is a Tumor-associated Immunomodulatory Ligand for CD33-related Siglecs

Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.     

Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR

Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling ³¹P NMR relaxation methods with spin-labeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are >1–2 μs (but <1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may be extended to a wide range of peripheral membrane proteins.     

Evaluation of Unrestrained Replica-Exchange Simulations Using Dynamic Walkers in Temperature Space for Protein Structure Refinement

A central problem of computational structural biology is the refinement of modeled protein structures taken from either comparative modeling or knowledge-based methods. Simulations are commonly used to achieve higher resolution of the structures at the all-atom level, yet methodologies that consistently yield accurate results remain elusive. In this work, we provide an assessment of an adaptive temperature-based replica exchange simulation method where the temperature clients dynamically walk in temperature space to enrich their population and exchanges near steep energetic barriers. This approach is compared to earlier work of applying the conventional method of static temperature clients to refine a dataset of conformational decoys. Our results show that, while an adaptive method has many theoretical advantages over a static distribution of client temperatures, only limited improvement was gained from this strategy in excursions of the downhill refinement regime leading to an increase in the fraction of native contacts. To illustrate the sampling differences between the two simulation methods, energy landscapes are presented along with their temperature client profiles.     

Quantitative Structure–Activity Relationship Model for Prediction of Protein–Peptide Interaction Binding Affinities between Human Amphiphysin-1 SH3 Domains and Their Peptide Ligands

Protein–protein interaction plays a critical role in signal transduction and many other key biological processes. The present study evaluated four parameters selected from among 554 physiochemical variables of 20 natural amino acids listed in AAindex, namely, hydrophobicity, electronic properties, steric properties, and hydrogen-bond properties. Human amphiphysin-1 Src homology 3 (SH3) domain-binding decapeptides were the object of analysis. A quantitative structure–activity relationship model of the SH3 domain-binding peptides was constructed using multivariate linear regression. The results showed that the four parameters ably characterize the structure of SH3 domain-binding decapeptides, have definitive physicochemical properties and a low level of computational complexity, are accessible, and may be used in integrated prediction models for other protein–peptide interactions.     

The Ileal Lipid Binding Protein Is Required for Efficient Absorption and Transport of Bile Acids in the Distal Portion of the Murine Small Intestine

The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6^−/− mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, P<0.05) in female but not male Fabp6^−/− mice. The activity of cholesterol 7α-hydroxylase (cyp7a1), the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, P<0.05) but not in male Fabp6^−/− mice. The amount of [³H]taurocholic acid (TCA) excreted by 24 h after oral administration was 102% (P<0.025) higher for female Fabp6^−/− mice whereas it was 57.3% (P<0.01) lower for male Fabp6^−/− mice, compared to wild-type mice. The retained fraction of the [³H]TCA localized in the small and large intestines was increased by 22% (P<0.02) and decreased by 62.7% (P<0.01), respectively, in male Fabp6^−/− mice relative wild-type mice, whereas no changes were seen in female Fabp6^−/− mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03) in both sexes of Fabp6^−/− mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.     

Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase

Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranslationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross-linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor.