Engineering and design of ligand-induced conformational change in proteins

The ability to manipulate ligand-induced conformational change, although representing a major challenge to the protein engineer, is an essential end point in efforts to produce novel functional proteins for biotechnology and therapeutic applications. Progress towards this goal requires determining not only what factors control the fold and stability of a protein, but also how ligand binding alters the complex conformational/energetic landscape. Important strides are being made on several fronts, including understanding the origin of long-range effects and allosteric structural mechanisms, using both experimental and theoretical approaches.     

Ligand specificity and ligand-induced conformational change in gal repressor

Gal repressor (GalR) binds D-galactose, which is responsible for lifting of repression of the gal operon. Proton T1 measurements of alpha- and beta-anomers of galactose as a function of gal repressor show preferential binding of the beta-anomer. The beta-anomer was isolated by high-performance liquid chromatography and was shown to bind tightly to GalR. Calorimetry was used to determine enthalpy changes at several temperatures. Heat capacity change was found to be positive, indicating that a significant amount of hydrophobic surface area was exposed upon galactose binding. Bis-ANS binding to GalR is significantly enhanced in the presence of a saturating amount of galactose, indicating additional exposure of hydrophobic surfaces. We propose that the galactose-induced conformational change involves the opening of the two subdomains, which may disrupt protein-protein interactions responsible for repression.     

Spectroscopic evidence for ligand-induced conformational change in NADP+:isocitrate dehydrogenase

Conformational changes induced by binding of ligands to cytosolic NADP(+)-specific isocitrate dehydrogenase from lactating bovine mammary gland were assessed using circular dichroism and fluorescence techniques. The secondary structure of isocitrate dehydrogenase, as monitored by CD spectra in the far-UV region, is unaltered by enzyme-ligand interactions; in contrast, dramatic changes occur in the near-UV region (270-290 nm) assigned to tyrosine and/or solvent-exposed tryptophan residues. Both the coenzyme analog, 2'-phosphoadenosine 5'-diphosphoribose, and NADPH have an effect on the CD spectrum which is opposite to that produced by metal complexes of either isocitrate or citrate. A CD band at 292 nm assigned to approximately 2 tryptophan residues in a hydrophobic environment is unchanged by binding of substrate or coenzyme. Approximately 30% of the intrinsic fluorescence of isocitrate dehydrogenase, corresponding to approximately 2 tryptophan residues, is not quenched by acrylamide in the absence of 6.3 M guanidine hydrochloride and remains unquenched in the enzyme-substrate complex. The constancy in the proportion of buried and exposed tryptophan residues implicates tyrosine in the observed near-UV CD spectral changes. Since binding of ligands does not influence quaternary structure (Seery, V.L., and Farrell, H. M., Jr. (1989) Arch. Biochem. Biophys. 274, 453-462), activation of isocitrate dehydrogenase may be related to a substrate-induced conformational transition.     

Allosteric regulation of a ribozyme activity through ligand-induced conformational change.

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.     

A ligand-induced conformational change in the Yersinia protein tyrosine phosphatase.

Protein tyrosine phosphatases (PTPases) play critical roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. The structures of several different PTPases have revealed a conserved active site architecture in which a phosphate-binding loop, together with an invariant arginine, cradle the phosphate of a phosphotyrosine substrate and poise it for nucleophilic attack by an invariant cysteine nucleophile. We previously reported that binding of tungstate to the Yop51 PTPase from Yersinia induced a loop conformational change that moved aspartic acid 356 into the active site, where it can function as a general acid. This is consistent with the aspartic acid donating a proton to the tyrosyl leaving group during the initial hydrolysis step. In this report, using a similar structure of the inactive Cys 403-->Ser mutant of the Yersinia PTPase complexed with sulfate, we detail the structural and functional details of this conformational change. In response to oxyanion binding, small perturbations occur in active site residues, especially Arg 409, and trigger the loop to close. Interestingly, the peptide bond following Asp 356 has flipped to ligate a buried, active site water molecule that also hydrogen bonds to the bound sulfate anion and two invariant glutamines. Loop closure also significantly decreases the solvent accessibility of the bound oxyanion and could effectively shield catalytic intermediates from phosphate acceptors other than water. We speculate that the intrinsic loop flexibility of different PTPases may be related to their catalytic rate and may play a role in the wide range of activities observed within this enzyme family.     

Activation and targeting of mitogen-activated protein kinases by G-protein-coupled receptors

Over the past decade, it has become apparent that many G-protein-coupled receptors (GPCRs) generate signals that control cellular differentiation and growth, including stimulation of Ras family GTPases and activation of mitogen-activated protein (MAP) kinase pathways. The mechanisms that GPCRs use to control the activity of MAP kinases vary between receptor and cell type but fall broadly into one of three categories: signals initiated by classical G protein effectors, e.g., protein kinase (PK)A and PKC, signals initiated by cross-talk between GPCRs and classical receptor tyrosine kinases, e.g., "transactivation" of epidermal growth factor (EGF) receptors, and signals initiated by direct interaction between beta-arrestins and components of the MAP kinase cascade, e.g., beta-arrestin "scaffolds". While each of these pathways results in increased cellular MAP kinase activity, emerging data suggest that they are not functionally redundant. MAP kinase activation occurring via PKC-dependent pathways and EGF receptor transactivation leads to nuclear translocation of the kinase and stimulates cell proliferation, while MAP kinase activation via beta-arrestin scaffolds primarily increases cytosolic kinase activity. By controlling the spatial and temporal distribution of MAP kinase activity within the cell, the consequences of GPCR-stimulated MAP kinase activation may be determined by the mechanism by which they are activated.     

Crystallographic complexes of surfactant protein A and carbohydrates reveal ligand-induced conformational change

Surfactant protein A (SP-A), a C-type lectin, plays an important role in innate lung host defense against inhaled pathogens. Crystallographic SP-A·ligand complexes have not been reported to date, limiting available molecular information about SP-A interactions with microbial surface components. This study describes crystal structures of calcium-dependent complexes of the C-terminal neck and carbohydrate recognition domain of SP-A with d-mannose, d-α-methylmannose, and glycerol, which represent subdomains of glycans on pathogen surfaces. Comparison of these complexes with the unliganded SP-A neck and carbohydrate recognition domain revealed an unexpected ligand-associated conformational change in the loop region surrounding the lectin site, one not previously reported for the lectin homologs SP-D and mannan-binding lectin. The net result of the conformational change is that the SP-A lectin site and the surrounding loop region become more compact. The Glu-202 side chain of unliganded SP-A extends out into the solvent and away from the calcium ion; however, in the complexes, the Glu-202 side chain translocates 12.8 Å to bind the calcium. The availability of Glu-202, together with positional changes involving water molecules, creates a more favorable hydrogen bonding environment for carbohydrate ligands. The Lys-203 side chain reorients as well, extending outward into the solvent in the complexes, thereby opening up a small cation-friendly cavity occupied by a sodium ion. Binding of this cation brings the large loop, which forms one wall of the lectin site, and the adjacent small loop closer together. The ability to undergo conformational changes may help SP-A adapt to different ligand classes, including microbial glycolipids and surfactant lipids.     

Strategies to identify ligands for orphan G-protein-coupled receptors

G-protein-coupled receptors are the most tractable class of protein targets for small molecule drug design. Sequencing of the human genome allied to bio-informatic analysis has identified a large number of putative receptors for which the natural ligands remain undefined. A range of currently employed and developing strategies to identify ligands that interact with these orphan receptors and to validate them as drug targets are described and discussed.     

A molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)-phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A. contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A. contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca(2+)-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase "active site" and the C-terminal "myotoxic site," justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.     

Conformation state-sensitive antibodies to G-protein-coupled receptors

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

Activation of the leptin receptor by a ligand-induced conformational change of constitutive receptor dimers

Binding of leptin to the leptin receptor is crucial for body weight and bone mass regulation in mammals. Leptin receptors were shown to exist as dimers, but the role of dimerization in receptor activation remains unknown. Using a quantitative Bioluminescence Resonance Energy Transfer approach, we show here in living cells that approximately 60% of the leptin receptor exists as constitutive dimers at physiological expression levels in the absence of leptin. No further increase in leptin receptor dimerization was detected in the presence of leptin. Importantly, in cells expressing the short leptin receptor isoform, leptin promoted a robust enhancement of energy transfer signals that reflect specific conformational changes of pre-existing leptin receptor dimers and that may be used as read-out in screening assays for leptin receptor ligands. Both leptin receptor dimerization and the leptin-induced energy transfer were Janus kinase 2-independent. Taken together, our data support a receptor activation model based on ligand-induced conformational changes rather than ligand-induced dimerization.     

Structure and ligand-induced conformational change of the 39-kDa glycoprotein from human articular chondrocytes

The 39-kDa human cartilage glycoprotein (HCGP39), a member of a novel family of chitinase-like lectins (Chilectins), is overexpressed in articular chondrocytes and certain cancers. Proposed functions of this protein include a role in connective tissue remodeling and defense against pathogens. Similar to other Chi-lectins, HCGP39 promotes the growth of connective tissue cells. The ability of HCGP39 to activate cytoplasmic signaling pathways suggests the presence of a ligand for this protein at the cell surface. There is currently no information regarding the identity of any physiological or pathological ligands of the Chi-lectins or the nature of the protein-ligand interaction. Here, we show that HCGP39 is able to bind chitooligosaccharides with micromolar affinity. Crystal structures of the native protein and a complex with GlcNAc8 show that the ligand is bound in identical fashion to family 18 chitinases. However, unlike the chitinases, binding of the oligosaccharide ligand to HCGP39 induces a large conformational change. Thus, HCGP39 could be a lectin that binds chitin-like oligosaccharide ligands and possibly plays a role in innate responses to chitinous pathogens, such as fungi and nematodes.     

Site-directed solid-state NMR measurement of a ligand-induced conformational change in the serine bacterial chemoreceptor

The challenging nature of studies of membrane proteins has made it difficult to determine the molecular mechanism of transmembrane signaling. For the bacterial chemoreceptor family, there are crystal structures of the internal and external domains, structural models of the transmembrane domain, and evidence for subtle ligand-induced conformational changes, but the signaling mechanism remains controversial. We have used a novel site-directed solid-state NMR distance measurement approach, using (13)C(19)F REDOR, to measure a ligand-induced change of 1.0 +/- 0.3 A in the distance between helices alpha 1 and alpha 4 of the ligand-binding domain in the intact, membrane-bound serine receptor. This distance change is shown not to be due to motion of the side chain and thus is due to motion of either the alpha 1 or the alpha 4 helix. Additional distance measurements can be used to determine the type of backbone motion and to follow it to the cytoplasm, to test and refine current proposals for the mechanism of transmembrane signaling. This is a promising general method for high-resolution measurements of local structure in intact, membrane-bound proteins.     

Prediction of a ligand-induced conformational change in the catalytic core of Cdc25A

The cell cycle control phosphatases Cdc25 are dual specificity phosphatases that dephosphorylate both phosphothreonine and phosphotyrosine residues on their substrate proteins. The determination of the apo-protein structure of Cdc25A revealed that this enzyme has a completely different fold compared to all other phosphatases crystallised to date. The conformation of the active site residues does not seem very suitable for catalysis in this unliganded structure. We have studied some structural features of the Cdc25A apo-structure and a modelled Cdc25A-ligand complex by molecular dynamics simulations. The simulations predict a conformational change in the peptide backbone of the complex, which is not observed in the apo-structure. This ligand-induced conformational change yields a structure that is similar to other protein tyrosine phosphatase-ligand complexes that have been crystallised. The change in conformation takes place in the position between a serine and a glutamic acid residue in the phosphate binding loop. We suggest that this type of conformational change is an important molecular switch in the catalytic process.     

Modelling G-protein-coupled receptors for drug design

The G-protein coupled receptors form a large and diverse multi-gene superfamily with many important physiological functions. As such, they have become important targets in pharmaceutical research. Molecular modelling and site-directed mutagenesis have played an important role in our increasing understanding of the structural basis of drug action at these receptors. Aspects of this understanding, how these techniques can be used within a drug-design programme, and remaining challenges for the future are reviewed.     

Probing the ligand-induced conformational change in HLA-DR1 by selective chemical modification and mass spectrometric mapping

Peptide binding induces conformational changes in class II MHC proteins that have been characterized using a variety of hydrodynamic and spectroscopic approaches, but these changes have not been clearly localized within the overall class II MHC structure. In this study, empty and peptide-loaded complexes of HLA-DR1, a common class II MHC variant, were chemically modified using the side chain-specific chemical modifiers p-hydroxyphenylglyoxal (arginine), tetranitromethane (tyrosine), N-bromosuccinimide (tryptpophan), and NHS-biotin (lysine). Modified proteins were subjected to in-gel digestion with trypsin and subsequent analysis by MALDI/MS. Three arginine residues and two lysine residues were differentially reactive, modified in the empty form but not the peptide-loaded form of the protein, indicating that the chemical reactivity of these regions differs in the two conformations. Three of the differential modifications were located on a single lateral face of the protein, indicating that this region is involved in the conformational change. Additionally, a number of lysine and tyrosine modification sites were present in both protein conformations. Overall, the pattern of reactivity is inconsistent with the idea that empty MHC molecules exist as molten globules or other partially unfolded intermediates, and suggests that the peptide-induced conformational change is localized to only a few regions of the protein.     

Modelling G-protein-coupled receptors for drug design.

The G-protein coupled receptors form a large and diverse multi-gene superfamily with many important physiological functions. As such, they have become important targets in pharmaceutical research. Molecular modelling and site-directed mutagenesis have played an important role in our increasing understanding of the structural basis of drug action at these receptors. Aspects of this understanding, how these techniques can be used within a drug-design programme, and remaining challenges for the future are reviewed.     

Classifying G-protein-coupled receptors to the finest subtype level

G-protein-coupled receptors (GPCRs) constitute a remarkable protein family of receptors that are involved in a broad range of biological processes. A large number of clinically used drugs elicit their biological effect via a GPCR. Thus, developing a reliable computational method for predicting the functional roles of GPCRs would be very useful in the pharmaceutical industry. Nowadays, researchers are more interested in functional roles of GPCRs at the finest subtype level. However, with the accumulation of many new protein sequences, none of the existing methods can completely classify these GPCRs to their finest subtype level. In this paper, a pioneer work was performed trying to resolve this problem by using a hierarchical classification method. The first level determines whether a query protein is a GPCR or a non-GPCR. If it is considered as a GPCR, it will be finally classified to its finest subtype level. GPCRs are characterized by 170 sequence-derived features encapsulating both amino acid composition and physicochemical features of proteins, and support vector machines are used as the classification engine. To test the performance of the present method, a non-redundant dataset was built which are organized at seven levels and covers more functional classes of GPCRs than existing datasets. The number of protein sequences in each level is 5956, 2978, 8079, 8680, 6477, 1580 and 214, respectively. By 5-fold cross-validation test, the overall accuracy of 99.56%, 93.96%, 82.81%, 85.93%, 94.1%, 95.38% and 92.06% were observed at each level. When compared with some previous methods, the present method achieved a consistently higher overall accuracy. The results demonstrate the power and effectiveness of the proposed method to accomplish the classification of GPCRs to the finest subtype level.