Knowledge-based model building of proteins: concepts and examples.

We describe how to build protein models from structural templates. Methods to identify structural similarities between proteins in cases of significant, moderate to low, or virtually absent sequence similarity are discussed. The detection and evaluation of structural relationships is emphasized as a central aspect of protein modeling, distinct from the more technical aspects of model building. Computational techniques to generate and complement comparative protein models are also reviewed. Two examples, P-selectin and gp39, are presented to illustrate the derivation of protein model structures and their use in experimental studies.     

Cloning,sequencing and expression of porcine CD40 ligand in Escherichia coli and human and porcine cells

The CD40L ligand (CD40L) plays an important role in the interaction between antigen-specific T lymphocytes and antigen-presenting cells. The porcine CD40L encoding gene was isolated from porcine peripheral blood mononuclear cells (PBMC) using RT-PCR. Sequence analysis of the cloned CD40L gene showed an open reading frame of 786 base pairs encoding a 262 amino acid protein with a predicted molecular mass of 29 kD. The deduced amino acid sequence of the porcine CD40L shared 82%, 88% and 93% similarity with the CD40L protein of mouse, human and cattle. The isolated CD40L sequence was expressed as a hexahistidine fusion protein in Escherichia coli and purified by affinity chromatography. The analysis of the CD40L-expression in human 293 and porcine MAX cells by immunofluorescence showed its location on the cell surface.     

Molecular model of the N-terminal receptor-binding domain of the human CD6 ligand ALCAM.

CD6-ligand interactions have been implicated in the regulation of T-cell adhesion and activation. CD6 is a member of the scavenger receptor family, whereas its human ligand (ALCAM) belongs to the immunoglobulin superfamily. The extracellular region of ALCAM includes five immunoglobulin-like domains. As a fusion protein, the N-terminal extracellular domain of ALCAM (ALCAMD1) binds specifically to CD6. We report the construction, assessment, and analysis of a molecular model of ALCAMD1. The model defines the CDR-analogous loops, the location of N-linked glycosylation sites, and residues that form the beta-sheet faces of the immunoglobulin-like domain. Predicted structural characteristics of the A'GFCC'C" face of the model are consistent with the presence of monomeric and dimeric forms of ALCAMD1, which has implications for the receptor-ligand interactions.     

The role of polar interactions in the molecular recognition of CD40L with its receptor CD40.

CD40 Ligand (CD40L) is transiently expressed on the surface of T-cells and binds to CD40, which is expressed on the surface of B-cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B-cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T-cell dependent B-cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X-ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X-ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, E114, E117). These residues were studied experimentally through site-directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, E114R, E117R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, E117N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge-reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74-R203, D84-R207, and E117-R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.     

A multimerized form of recombinant human CD40 ligand supports long-term activation and proliferation of B cells

Background aimsCD40-activated B cells have long been studied as potent antigen-presenting cells that can potentially be used for cancer immunotherapy. Nevertheless, their use in human clinical trials has been limited by the lack of a Good Manufacturing Practice–grade soluble human CD40 ligand that is able to induce activation and proliferation of primary B cells. We describe an in vitro method to effectively generate and expand B cells through the use of a multimerized form of human recombinant CD40 ligand (rCD40L).MethodsHuman B cells were isolated from healthy donors and cultivated with either rCD40L or on a monolayer of murine NIH3T3 cells stably expressing human CD40L (NIH3T3/tCD40L) as a widely used standard method. Morphology, expansion rate, immune phenotype and antigen presentation function were assessed.ResultsB cells efficiently proliferated in response to rCD40L over 14 days of culture in comparable amounts to NIH3T3/tCD40L. B-cell division in response to CD40L was also confirmed by carboxyfluorescein succinimidyl ester dilution. Moreover, rCD40L induced on B cells upregulation of co-stimulatory molecules essential for antigen presentation. Additionally, proliferation of T cells from allogeneic healthy volunteers confirmed the immunostimulatory capacities of CD40-activated B cells.ConclusionsWe demonstrated that B cells with potent antigen presentation capacity can be generated and expanded by use of a non-xenogeneic form of CD40L that could be implemented in future human clinical settings.     

Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.     

Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser¹³² loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.     

Programmed cell death: the influence of CD40, CD95 (Fas or Apo-I) and their ligands

Programmed cell death (PCD) or apoptosis is a process whereby developmental or environmental stimuli activate a specific series of events that culminate in cell death. PCD is essential for normal development and abnormality in the process can lead to defects ranging from embryonic lethality and tissue-specific perturbation of postnatal development to a high susceptibility to malignancy. Therapeutics that modulate the regulation of PCD may provide a new opportunity for the treatment of the PCD related diseases and cancer. CD40 and CD95 (Fas/Apo-I) are transmembrane proteins of the nerve growth factor/tumour necrosis factor α receptor superfamily. The death signal of PCD occurs when the CD95 receptor on the cell surface binds to the CD95 ligand (CD95L) or to the anti-CD95 monoclonal antibody (mAb). In contrast, PCD could be inhibited by the survival signal mediated from the binding of the CD40 receptor to the CD40 ligand (CD40L) or to the anti-CD40 mAb. In this review, the interaction of CD40/CD40L and CD95/CD95L on PCD in normal and malignant cells is discussed.     

Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene

Genetic material obtained from various individuals may contain certain polymorphisms which may conflict with the predetermined DNA sequence and consequently, may modulate the function of gene products. In this study, coding sequence of rat CD40 ligand (CD40L, CD154) was obtained from activated splenocytes, amplified, and cloned into a eukaryotic expression vector by using directional cloning method. Sequence of the recombinant rat CD40L DNA, pCD40L-IRES2-EGFP (pCD40L), was compared with the previously reported rat CD40L cDNA sequences and a 99% identity was found. Differing nucleotides were on the positions; 122-T/C, 341-G/A, 476-G/A, 762-T/A. Further alignment analysis showed that pCD40L was collectively carrying the nucleotides each previously reported by different groups. The sequence was submitted to NCBI GenBank and nucleotide database accession number EF066490 was obtained. Following transfection of the construct into NIH/3T3 cell line, novel CD40L clone was functionally expressed de novo, increasing the expression of CD80 and CD86 costimulatory molecules and augmenting the proliferation rate of effector splenocytes in immune reactions ex vivo. Based on these data, here we report a novel recombinant clone of the rat CD40L gene which may represent a potential polymorphic variant.     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.     

Epstein Barr virus inhibits the stimulatory effect of TLR7/8 and TLR9 agonists but not CD40 ligand in human B lymphocytes

Viruses and other microorganisms express specific pathogen‐associated molecular patterns that are recognized by cell surface or endosome‐associated Toll‐like receptors (TLR). There are many examples of viruses that have developed strategies to modulate TLR signaling through the use of viral or cellular molecules. Epstein–Barr virus (EBV) has recently been found to display a complex interaction with TLR. The aim of this study was to asses the effect of EBV infection on proliferative capacity of TLR7/8 and 9 agonist and CD40 ligand (CD40L) in normal B lymphocytes. Our results demonstrate that EBV induces a significant inhibition in proliferative response to TLR7/8 (P < 0.004) and TLR9 (P < 0.000) agonists but not to CD40L stimulation in enriched human normal B lymphocytes. Similar inhibitory effect was also observed in B lymphocytes prestimulated with the TLR agonists, implying that the suppressive effect is not due to downregulation of TLR protein expression by EBV. EBV infection did not induce apoptosis and did not downregulate TLR7/8 mRNA expression in B lymphocytes. Our results suggest that EBV might be able to evade the immune system by modulation of the TLR signaling pathway.     

Parametric sensitivity analysis and reduction of a detailed nutritional model of plant cell cultures

A kinetic model of plant nutrition described by Cloutier et al. (Cloutier et al., 2008. Biotechnol Bioeng 99:189-200) is progressively simplified so as to obtain a predictive model that describes the evolution of the biomass and the extracellular and intracellular concentrations of three determining nutrients, that is, free intracellular nitrogen, phosphate, and carbohydrate compounds. Three techniques of global sensitivity analysis are successively applied to assess the model parameter influence and potential correlation. The resulting dynamic model is able to predict plant growth for the two most encountered plant bioprocesses, namely suspension cells and hairy roots.     

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.     

Molecular Modeling of Immunoglobulin Superfamily Proteins: CTLA-4 (CD152) - Comparison of a Predicted and Experimentally Determined Three-Dimensional Structure

The CD28 and CTLA-4 (CD152) receptors on T cells recognize CD80 and CD86 ligands on antigen presenting cells. These interactions provide and control costimulatory signals required for effective T cell activation. CD28 and CTLA-4 belong to the immunoglobulin superfamily (IgSF) and contain a single extracellular ligand binding domain. The three-dimensional (3D) structure of the binding domain of CTLA-4 was modeled previously using a combination of structure-based sequence comparison, IgSF consensus residue analysis, conformational search, and inverse folding calculations. Recently, the 3D structure of CTLA-4 was determined by NMR. Comparison of the modeled and experimentally determined CTLA-4 structure has made it possible to assess the accuracy of our predictions. We found that the overall accuracy of the model was sound and sufficient for a meaningful application of the model in experimental studies. Major errors in the model are limited to the conformation and position of some loops. Our studies on CTLA-4 provide an example for the opportunities and limitations of comparative protein modeling in the presence of low sequence similarity.Electronic Supplementary Material available.     

Structure-based modeling of the ligand binding domain of the human cell surface receptor CD23 and comparison of two independently derived molecular models.

CD23, a type II membrane receptor protein, recognizes four different ligands via its extracellular C-type lectin domain: immunoglobulin E (IgE), CD21, and the beta 2-integrins CD11b and CD11c. CD23 specifically interacts in a calcium-dependent manner, "lectin-like" with carbohydrate moieties expressed on CD21 and CD11b/c, but also "lectin-unlike" with protein epitopes on IgE. As a first step in analyzing the multiple binding specificities associated with CD23 in more detail, we report a detailed molecular model of the lectin-like domain of human CD23 (hCD23). The model was built based on information provided by X-ray structures of mannose binding protein (MBP) and E-selectin, both of which are members of the calcium-dependent (C-type) lectin superfamily. Sequence-structure comparisons suggest that hCD23 is structurally more similar to MBP than to E-selectin. The hCD23 model is compared to an independently derived model. Although the CD23-carbohydrate and CD23-protein interactions are both calcium dependent, analysis of the model suggests the presence of distinct binding sites for these ligands.     

Structure of segments of a G protein-coupled receptor: CD and NMR analysis of the saccharomyces cerevisiae tridecapeptide pheromone receptor

Peptides representing both loop and the sixth transmembrane regions of the α-factor receptor of Saccharomyces cerevisiae were synthesized by solid-phase procedures and purified to near homogeneity. CD, nmr, and modeling analysis indicated that in aqueous media the first extracellular loop peptide E1(107–125), the third intracellular loop peptide I3(231–243), and the carboxyl terminus peptide I4(350–372) were mostly disordered. In contrast, the second extracellular loop peptide E2(191–206) assumed a well-defined structure in aqueous medium and the sixth transmembrane domain peptide receptor M6(252-269, C252A) was highly helical in trifluoroethanol/water (4:1), exhibiting a kink at Pro258. A synthetic peptide containing a sequence similar to that of the sixth transmembrane domain of a constitutively active α-factor receptor M6(252–269, C252A, P258L) in which Leu replaces Pro258 exhibited significantly different biophysical properties than the wild-type sequence. In particular, this peptide had very low solubility and gave CD resembling that of a β-sheet structure in hexafluoroacetone/water (1:1) whereas the wild-type peptide was partially helical under identical conditions. These results would be consistent with the hypothesis that the constitutive activity of the mutant receptor is linked to a conformational change in the sixth transmembrane domain. The study of the receptor segments also indicate that peptides corresponding to loops of the α-factor receptor do not appear to assume turn structures. © 1998 John Wiley & Sons, Inc. Biopoly 46: 343–357, 1998     

Assessment of the effect of TLR7/8, TLR9 agonists and CD40 ligand on the transformation efficiency of Epstein-Barr virus in human B lymphocytes by limiting dilution assay

Infection of human B cells with Epstein-Barr virus (EBV) induces polyclonal activation in almost all infected cells, but a small proportion of infected cells are transformed to immortalized lymphoblastoid cell lines. Since B cells are activated also by CD40 ligand (CD40L) and Toll-like receptor (TLR) agonists via a similar signaling pathway, it is likely that costimulation through these molecules could result in synergistic enhancement of the transformation efficiency of EBV. In this study, the stimulatory effect of TLR7/8 (R848), TLR9 (CpG) agonists and/or CD40L on transformation efficiency of EBV in normal human B cells was assessed using the limiting dilution assay. Costimulation of peripheral blood mononuclear cells (PBMCs) with CpG and R848, but not CD40L, increased significantly the frequency of EBV transformed B cells (p < 0.001). Neither synergistic nor additive effects were observed between TLR agonists and CD40L and also TLR7/8 and TLR9 agonists. Costimulation with R848, CpG and CD40L enhanced the proliferative response of B cells infected with EBV. This effect was more evident when enriched B cells were employed, compared to PBMCs. The promoting effect of TLR agonists stimulation, implies that EBV may take advantage of the genes induced by the TLR stimulation pathway for viral latency and oncogenesis.