整联蛋白与其配体结合的分子机制研究进展

整联蛋白是细胞表面的主要膜受体,具有介导细胞与细胞基质间的黏附及病毒吸附细胞等功能。αⅠ结构域型整联蛋白和非αⅠ结构域型整联蛋白与配体结合的方式各异。对整联蛋白-配体复合物分子结构的研究表明,整联蛋白利用其配体结合位点的αMIDAS（依赖金属离子的吸附位点）、βMIDAS与各种类型配体结合。     

Use of phage display to probe the evolution of binding specificity and affinity in integrins

The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.     

A Host-Guest Relationship in Bone Morphogenetic Protein Receptor-II Defines Specificity in Ligand-Receptor Recognition

One of the most intriguing questions confronting the bone morphogenetic protein family is the mechanism of ligand recognition, because there are more ligands than receptors. Crystal structures of two type II receptors, ActR-II and BMPR-II, are essentially identical, and a loop structure (A-loop) has been suggested to play a role in determining ligand specificity. A solution biophysical study showed mutations of several A-loop residues in these two receptors exert different ligand binding effects. Thus, the issues of mechanism of ligand recognition and specificity remain unresolved. We examined effects of mutations of residues Y40, G47, and S107 in BMPR-II. These residues are not identified as being in contact with the ligand in the BMP-7-BMPR-II complex but are found mutated in genetic diseases. They are likely to be useful in identifying their roles in differentiating the various BMP ligands. Spectroscopic probing revealed little mutation-induced structural change in BMPR-II. Ligand binding studies revealed that Y40 plays a significant role in differentiating three distinct ligands; G47 and S107 affect ligand binding to a lesser extent. The role of the A-loop in ActR-II or BMPR-II is dependent on the host sequence of the receptor extracellular domain (ECD) in which it is embedded, suggesting a host-guest relationship between the A-loop and the rest of the ECD. Computational analysis demonstrated a long-range connectivity between Y40, G47, and S107 and other locations in BMPR-II. An integration of these results on functional energetics and protein structures clearly demonstrates, for the first time, an intradomain communication network within BMPR-II.     

甾体激素受体功能特异性的结构基础

甾体激素受体家族包括雌激素受体、雄激素受体等五个亚家族,在机体组织细胞的生长分化、发育生殖、内环境稳定等几乎所有生理过程中都起着重要的作用。研究甾体激素受体亚家族的特异性可以加深对该家族功能的理解,并且具有潜在的临床应用价值。采用进化踪迹方法对该家族的配体结合域（LBD）进行分析,探讨了决定亚家族功能特异性的结构基础。结果表明,甾体激素受体的各亚家族可能同相应的内源性配体存在着共进化关系;配体结合处的踪迹残基决定了受体－配体间的氢键作用和疏水相互作用模式并导致了亚家族的配体结合特异性。上述结论可用于甾体激素受体的配体结合特异性的改造以及新型组织选择性配体（如选择性雌激素受体调节剂,SERM）的设计。     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Theoretical Study of the Sequence Selectivity of Isolexins,Isohelical DNA Groove Binding Ligands. Proposal for the GC Minor Groove Specific Compounds

Abstract

Atheoretical study is presented of complex formation between DNA fragments of different base sequences and isolexins, “isohelical base reading polymers”, formed of heteroaromatic pentagonal rings joined by appropriate linkers. Extensive computations are performed for the isolexin composed of the furan-pyrrole-furan sequence. They involve charged ligands with propioamidinium groups at both ends as well eis neutral molecules with terminal methyl, carbonyl and amino groups. Two different groups (C=O and NH) are used as linkers between the base reading moieties. The role of these elements on the binding preference of the ligands has been examined. The results show that the mere possibility of formation of hydrogen bonds between a ligand and the nucleic acid bases is not sufficient to ensure its binding specificity which is determined largely by the interplay of electrostatic factors. Thus the dicationic isolexins uniformly prefer AT sequences. For the neutral isolexins the nature of the groups forming the linkers is a major factor in defining the specificity, although these groups do not participate directly in the interaction with DNA The C=O linkers favour binding to AT sequence while the N-H linkers permit preferential binding to the GAG sequence. Finally, for the first time in theoretical computations, a ligand is proposed which should bind preferentially to the minor groove of GC sequences: this ligand is a neutral isolexin composed of three furan rings linked by two N-H groups. This ligand is considered as an improvable prototype. Altogether the results presented open the path for the designing of minor groove ligands specific for any desirable DNA base sequence.     

Characteristics of fibrinogen binding to the domain of CD11c, an alpha subunit of p150,95.

beta2 integrins on leukocytes play important roles on cell-cell or cell-matrix adhesion through their ability to bind multiple ligands. The alpha subunits of leukocyte CD11/CD18 integrins contain an approximately 200-amino-acid inserted domain (I-domain) which is implicated in ligand binding function. To understand the characteristics of ligand binding to the alpha subunit of beta2 integrin p150,95 (CD11c/CD18), a recombinant form of the I-domain of CD11c was generated and analyzed for the interaction with fibrinogen, one of the ligands of p150,95. It was found that the CD11c I-domain bound fibrinogen specifically. Fibrinogen binding to the CD11c I-domain was inhibited by a molar excess of fragment E, a central domain of fibrinogen, and not by that of fragment D, a distal domain of fibrinogen, suggesting that CD11c/CD18 recognizes a central domain of fibrinogen. Divalent cations such as Mg(2+) and Mn(2+) were required for fibrinogen binding to the CD11c I-domain. Also alanine substitutions on the putative metal binding sites of the CD11c I-domain such as Asp(242) and Tyr(209) reduced its ability to bind fibrinogen. These data reinforce the fact that the divalent cation is a prerequisite for ligand binding of the CD11c I-domain.     

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.     

Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling

How ligand binding alters integrin conformation in outside-in signaling, and how inside-out signals alter integrin affinity for ligand, have been mysterious. We address this with electron microscopy, physicochemical measurements, mutational introduction of disulfides, and ligand binding to alphaVbeta3 and alphaIIbbeta3 integrins. We show that a highly bent integrin conformation is physiological and has low affinity for biological ligands. Addition of a high affinity ligand mimetic peptide or Mn(2+) results in a switchblade-like opening to an extended structure. An outward swing of the hybrid domain at its junction with the I-like domain shows conformational change within the headpiece that is linked to ligand binding. Breakage of a C-terminal clasp between the alpha and beta subunits enhances Mn(2+)-induced unbending and ligand binding.     

Computational Study of Synthetic Agonist Ligands of Ionotropic Glutamate Receptors

Neurological glutamate receptors are among the most important and intensely studied protein ligand binding systems in humans. They are crucial for the functioning of the central nervous system and involved in a variety of pathologies. Apart from the neurotransmitter glutamate, several artificial, agonistic and antagonistic ligands are known. Of particular interest here are novel photoswitchable agonists that would open the field of optogenetics to glutamate receptors. The receptor proteins are complex, membrane-bound multidomain oligomers that undergo large scale functional conformational changes, making detailed studies of their atomic structure challenging. Therefore, a thorough understanding of the microscopic details of ligand binding and receptor activation remains elusive in many cases. This topic has been successfully addressed by theoretical studies in the past and in this paper, we present extensive molecular dynamics simulation and free energy calculation results on the binding of AMPA and an AMPA derivative, which is the basis for designing light-sensitive ligands. We provide a two-step model for ligand binding domain activation and predict binding free energies for novel compounds in good agreement to experimental observations.     

Structural basis of non-specific lipid binding in maize lipid-transfer protein complexes revealed by high-resolution X-ray crystallography

Non-specific lipid-transfer proteins (nsLTPs) are involved in the movement of phospholipids, glycolipids, fatty acids, and steroids between membranes. Several structures of plant nsLTPs have been determined both by X-ray crystallography and nuclear magnetic resonance. However, the detailed structural basis of the non-specific binding of hydrophobic ligands by nsLTPs is still poorly understood. In order to gain a better understanding of the structural basis of the non-specific binding of hydrophobic ligands by nsLTPs and to investigate the plasticity of the fatty acid binding cavity in nsLTPs, seven high-resolution (between 1.3 A and 1.9 A) crystal structures have been determined. These depict the nsLTP from maize seedlings in complex with an array of fatty acids.A detailed comparison of the structures of maize nsLTP in complex with various ligands reveals a new binding mode in an nsLTP-oleate complex which has not been seen before. Furthermore, in the caprate complex, the ligand binds to the protein cavity in two orientations with equal occupancy. The volume of the hydrophobic cavity in the nsLTP from maize shows some variation depending on the size of the bound ligands.The structural plasticity of the ligand binding cavity and the predominant involvement of non-specific van der Waals interactions with the hydrophobic tail of the ligands provide a structural explanation for the non-specificity of maize nsLTP. The hydrophobic cavity accommodates various ligands from C10 to C18. The C18:1 ricinoleate with its hydroxyl group hydrogen bonding to Ala68 possibly mimics cutin monomer binding which is of biological importance. Some of the myristate binding sites in human serum albumin resemble the maize nsLTP, implying the importance of a helical bundle in accommodating the non-specific binding of fatty acids.     

The roles of contact residue disorder and domain composition in characterizing protein-ligand binding specificity and promiscuity

Most protein chains interact with only one ligand but a small number of protein chains can bind several ligands, and many examples are available in the protein-ligand complex database of PDB. Among these proteins, some show preferences for the ligands or types of ligands they bind; however, so far we have only poor understanding of what determines protein-ligand binding and its specificity. Here we investigate the structural and functional properties of proteins in protein-ligand complexes. Analysis of the protein-ligand complex dataset from the PDB structure database reveals that proteins with more interactions have more disordered contact residues. Those proteins containing few disordered contact residues that bind multiple ligands have a tendency to consist of several domains. Analysis of physicochemical properties of hub contact residues binding multiple ligands indicates that they are enriched for hydrophilic, charged, polar and His-Asp catalytic triad residues. Finally, in order to differentiate proteins binding different classes of ligands, we mapped the three most prominent classes of ligands onto different superfamily domains. Our results demonstrate that contact residue disorder and ordered multiple domains are complementary factors that play a crucial role in determining ligand binding specificity and promiscuity.     

Entropy and Mg2+ control ligand affinity and specificity in the malachite green binding RNA aptamer

The binding of small molecule targets by RNA aptamers provides an excellent model to study the versatility of RNA function. The malachite green aptamer binds and recognizes its ligand via stacking and electrostatic interactions. The binding of the aptamer to its original selection target and three related molecules was determined by isothermal titration calorimetry, equilibrium dialysis, and fluorescence titration. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. These data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands whereas the complex with the original selection target is stable at low salt and in the absence of divalent metal ions.     

Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1

The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.     

Cation-pi interactions as determinants for binding of the compatible solutes glycine betaine and proline betaine by the periplasmic ligand-binding protein ProX from Escherichia coli

Compatible solutes such as glycine betaine and proline betaine are accumulated to exceedingly high intracellular levels by many organisms in response to high osmolarity to offset the loss of cell water. They are excluded from the immediate hydration shell of proteins and thereby stabilize their native structure. Despite their exclusion from protein surfaces, the periplasmic ligand-binding protein ProX from the Escherichia coli ATP-binding cassette transport system ProU binds the compatible solutes glycine betaine and proline betaine with high affinity and specificity. To understand the mechanism of compatible solute binding, we determined the high resolution structure of ProX in complex with its ligands glycine betaine and proline betaine. This crystallographic study revealed that cation-pi interactions between the positive charge of the quaternary amine of the ligands and three tryptophan residues forming a rectangular aromatic box are the key determinants of the high affinity binding of compatible solutes by ProX. The structural analysis was combined with site-directed mutagenesis of the ligand binding pocket to estimate the contributions of the tryptophan residues involved in binding.     

The integrins

The integrins are a superfamily of cell adhesion receptors that bind to extracellular matrix ligands, cell-surface ligands, and soluble ligands. They are transmembrane αβ heterodimers and at least 18 α and eight β subunits are known in humans, generating 24 heterodimers. Members of this family have been found in mammals, chicken and zebrafish, as well as lower eukaryotes, including sponges, the nematode Caenorhabditis elegans (two α and one β subunits, generating two integrins) and the fruitfly Drosophila melanogaster (five α and one β, generating five integrins). The α and β subunits have distinct domain structures, with extracellular domains from each subunit contributing to the ligand-binding site of the heterodimer. The sequence arginine-glycine-aspartic acid (RGD) was identified as a general integrin-binding motif, but individual integrins are also specific for particular protein ligands. Immunologically important integrin ligands are the intercellular adhesion molecules (ICAMs), immunoglobulin superfamily members present on inflamed endothelium and antigen-presenting cells. On ligand binding, integrins transduce signals into the cell interior; they can also receive intracellular signals that regulate their ligand-binding affinity. Here we provide a brief overview that concentrates mostly on the organization, structure and function of mammalian integrins, which have been more extensively studied than integrins in other organisms.     

Determinants of ligand binding specificity in the glial cell line-derived neurotrophic factor family receptor alpha S

The glial cell line-derived neurotrophic factor (GDNF) family comprise a subclass of cystine-knot superfamily ligands that interact with a multisubunit receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich glycosyl phosphatidylinositol-anchored binding subunit called GDNF family receptor alpha (GFRalpha). All four GDNF family ligands utilize c-Ret as a common signaling receptor, whereas specificity is conferred by differential binding to four distinct GFRalpha homologues. To understand how the different GFRalphas discriminate ligands, we have constructed a large set of chimeric and truncated receptors and analyzed their ligand binding and signaling capabilities. The major determinant of ligand binding was found in the most conserved region of the molecule, a central domain predicted to contain four conserved alpha helices and two beta strands. Distinct hydrophobic and positively charged residues in this central region were required for binding of GFRalpha1 to GDNF. Interaction of GFRalpha1 and GFRalpha2 with GDNF and neurturin required distinct subsegments within this central domain, which allowed the construction of chimeric receptors that responded equally well to both ligands. C-terminal segments adjacent to the central domain are necessary and have modulatory function in ligand binding. In contrast, the N-terminal domain was dispensable without compromising ligand binding specificity. Ligand-independent interaction with c-Ret also resides in the central domain of GFRalpha1, albeit within a distinct and smaller region than that required for ligand binding. Our results indicate that the central region of this class of receptors constitutes a novel binding domain for cystine-knot superfamily ligands.     

Contributions by ionic and steric features of ligands to their binding with phosphorylcholine-specific immunoglobulin IgA H-8 as determined by fluorescence spectroscopy.

The murine myeloma IgA H-8 Fab' fragment which exhibits a binding specificity for phosphorylcholine was assayed for its ability to bind with a number of charged ligands. Monitoring of the ligand-induced changes of protein fluorescence provided a fast and accurate method of determining the equilibrium binding constants. The binding data along with fluorescence spectral properties of the protein permitted an assessment of the relative importance of some binding parameters as well as an evaluation of certain ionic and steric contributions made by ligands exhibiting significant binding affinity for the antibody fragment. Among the conclusions reached is that the dielectric of the binding site microenvironment is important in determining the strength of binding and that hydrophobic groups surrounding a quaternary cationic ligand are important in creating an appropriate binding site of low dielectric value.     

Urokinase-type plasminogen activator receptor (CD87) is a ligand for integrins and mediates cell-cell interaction

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.