Molecular modelling of UH-301 and 5-HTla receptor interactions

A three-dimensional model of the human 5-HT_1a receptor was constructedby molecular modelling, and the molecular and electronic structuresof (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin(UH-301) and of (R)- and (S)-8-hydroxy-2-(dipropylamino)tetralin(8-OH-DPAT) were examined by molecular mechanics and quantummechanics calculations and molecular dynamics simulations. Thereceptor model has seven transmembrane helices (TMHs), organizedaccording to a projection map of visual rhodopsin, and includesall loops between helices and the N- and C-terminal parts. Interactionsof UH-301 and 8-OH-DPAT with the 5-HT_1a receptor were examinedby molecular dynamics simulations and energy minimization ofreceptor–ligand complexes. 8-OH-DPAT had lower electrostaticpotentials around the hydroxyl group and stronger hydrogen bondingto the receptor model than had UH-301. The simulations indicatedthat the 5-HT_1a receptor agonists, (R)- and (S)-8-OH-DPAT and(R)-UH-301, interacted with the receptor at a site closer toAsp82 in TMH2 than did (S)-UH-301, which is a 5-HT,_1a receptorantagonist. Simulations of receptor–ligand complexes indicatedthat Asp82, Asp116, Ser199, Thr200 and De385 are essential forbinding of both agonist and antagonist to the receptor.     

Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9

Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.     

Structural characterization by computer experiments of the lipid-free LDL-receptor-binding domain of apolipoprotein E

The structure and dynamics of the lipid-free LDL-receptor-bindingdomain of apolipoprotein E (apoE-RBD) has been investigatedby Molecular Dynamics Simulations. ApoE-RBD in its monomericlipid-free form is a singular four-helix bundle made up of fourelongated amphipathic helices. Analysis of one 1.5 ns moleculardynamics trajectory of apoE-RBD performed in water indicatesthat the lipid-free domain adopts a structure that exhibitscharacteristics found in native proteins: it has very stablehelices and presents a compact structure. Yet its interior exhibitsa larger number of transient atomic-size cavities relative tothat found in other proteins of similar size and its apolarside chains are more mobile. The latter features distinguishthe elongated four-helix bundle as a slightly disordered structure,which shows a structural likeness with some de novo designedfour-helix bundle proteins and shares with the latter a leucine-richresidue composition. We anticipate that these unique propertiescompared with other native helix bundles may be related to thepostulated ability of apoE-RBD to undergo an opening of itsbundle upon interaction with phospholipids. The distributionof empty cavities computed along the trajectory in the interfaceregions between the different pairs of helices reveals thatthe tertiary contacts in one of the interfaces are weaker suggestingthat this particular interface could be more easily rupturedupon lipid association.     

The Effect of a Protein Environment on the Proposed Activation Mechanism of the Histamine H2-Receptor

Lack of information on the molecular structures of receptor proteins hampers the development of an understanding of their mechanisms. Small molecular models have served in the study of discrete details of such mechanisms, but receptor processes like recognition and activation take place in the protein environment of the receptor which may have a role in the process. To learn about the possible role of the protein environment in these mechanisms, we followed a heuristic approach in which models are used that range from small molecular complexes to macromolecular environments that model the receptors. The underlying assumption in such modelling is that similar elements of secondary structure, present in the model proteins as well as in the receptors, will make similar contributions to the molecular mechanism in which they function. We report the results of computational simulations of the activation mechanisms in a model protein environment used to study the effect of the protein structure on the proposed activation mechanism of the H₂-receptor of the neurotransmitter histamine. The Protein Data Bank was searched for a protein of known structure that could serve as a model for a macromolecular environment of the histamine H₂-receptor, based on a series of requirements defined by findings from our previous work on small molecular complexes modelling recognition and activation mechanisms in that receptor. The enzyme citrate synthase, E.C. 4.1.3.7, was found to contain the structural elements necessary for such a model. The activation mechanism simulated inside this protein consists of two proton transfer steps: one from the receptor to the neurotransmitter and another from it to a different site of the receptor. Quantum chemical methods were used to calculate the energetics of the proton transfers inside the protein structure represented as a collection of point charges and polarizable dipoles included in the Hamiltonian. The energetic contribution from polarization, as well as the interaction of the proton transfer system with the helix dipoles, is found to affect the proton transfer mechanism. The results suggest protein engineering approaches that can be used to modulate receptor activity by specific changes in the protein structure.     

A low resolution model for the interaction of G proteins with G protein-coupled receptors

A model is presented for the interaction between G proteinsand G protein-coupled receptors. The model is based on the factthat this interaction shows little specificity and thus conservedparts of the G proteins have to interact with conserved partsof the receptors. These parts are a conserved negative residuein the G protein, a fully conserved arginine in the receptorand a series of residues that are not conserved but always hydrophobiclike the hydrophobic side of the C-terminal helix of the G proteinand the hydrophobic side of a helix in the C-terminal domainof the receptor. Other, mainly cytosolic, factors determinethe specificity and regulation of this interaction. The relationbetween binding and activation will be shown. A large body ofexperimental evidence supports this model. Despite the factthat the model does not provide atomic resolution, it can beused to explain some experimental data that would otherwiseseem inexplicable, and it suggests experiments for its falsificationor verification.     

Sequence divergence analysis for the prediction of seven-helix membrane protein structures: I. Comparison with bacteriorhodopsin

A method using protein sequence divergence to predict the three-dimensionalstructure of the transmembrane domain of seven-helix membraneproteins is described. The key component in the multistep procedureis the calculation of a hydrophilic and lipophilic variabilityindex for each amino acid in an alignment of a family of homologousproteins. The variability profile, a plot of the calculatedvariability index versus alignment position, can be used topredict a tertiary model of the backbone conformation of thetransmembrane domain. This method was applied to bacteriorhodopsin(BR) and the model obtained was compared with the known structureof this protein. Using an alignment of the amino acid sequencesof BR and closely related (20% identity) proteins, the boundariesof the transmembrane regions, their secondary structures andorientations inside the membrane bilayer were predicted basedon the variability profile. Additional information about theshape of the helix bundle was also obtained from the averagevariability of each transmembrane helix with the assumptionthat the helices are packed sequentially and form a closed helixbundle. Correct features of the known structure of BR were foundin the model structure, suggesting that a similar strategy canbe used to predict transmembrane helices and the packing shapeof other membrane proteins with seven transmembrane helices,such as the opsins and other G-protein coupled receptors.     

A model for the C5a receptor and for T its interaction with the ligand

A model of the C5a receptor was built based on the assumptionthat the seven membrane-spanning helices of known inward/outwarddirection are in an arrangement roughly similar to that in bacteriorhodopsin.Guidelines for the positioning of the helices were cysteinepairing, ‘ridges into grooves’ interdigitation ofside chains and aromatic cluster formation. The chain segmentsprotruding from the membrane are too short for folding intoan independent ectodomain. The only longer segment (179–202)is tied down in its centre onto the membrane by a disulphidebridge and, thereby, made into two short loops as well. Ideasof the interaction of the C5a receptor with its ligand werederived mainly from the search for accommodation of the functionallyessential arginine residues 40 and 74 of C5a. Asp82 is the onlycharged residue in a pocket {small tilde}20 A below the receptorsurface and is conserved in the rhodopsin superfamily. It commendsitself for binding Arg74 which is the tip of the flexible C-terminalchain of C5a, and rules out Arg40 in the structurally well-definedpart of the molecule. The latter may bind to Glul80 at the bottomof a more shallow pocket which happens to resemble the substrate-bindingsite of trypsin.     

A 3D model of the {delta} opioid receptor and ligand-receptor complexes

A model for the 3D structure of the transmembrane domain ofthe opioid receptor was predicted from the sequence divergenceanalysis of 42 sequences of G-protein coupled peptide hormonereceptors belonging to the opioid, somatostatin and angiotensinreceptor families. No template was used in the prediction steps,which include multiple sequence alignment, calculation of avariability profile of the aligned sequences, use of the variabilityprofile to identify the boundaries of transmembrane regions,prediction of their secondary structure, optimization of thepacking shape in a helix bundle, prediction of side chain conformationsand structural refinement The general shape of the model issimilar to that of the low resolution rhodopsin structure inthat the TM3 and TM7 helices are most buried in the bundle andthe TM1 and TM4 helices are most exposed to the lipid phase.An initial assessment of this model was made by determiningto what extent a binding site identified using four structurallydisparate high affinity opioid ligands was consistent withknown mutational studies. With the assumption that the pro-tonatedamine nitrogen, a feature common to all opioid ligands, interactswith the highly conserved Aspl27 in TM3, a pocket was foundthat satisfied the criteria of complementarity to the requirementsfor receptor recognition for these four diverse ligands, two selective antagonists (the fused ring naltrindole and the peptideTyr-Tic-Phe-Phe-NH₂) and the two agonists lofentanil and BW373U86deduced from previous studies of the ligands alone. These ligandscould be accommodated in a similar region of the receptor. Thereceptor binding site identified in the optimized complexescontained many residues in positions known to affect ligandbinding in G-protein coupled receptors. These results also allowedidentification of key residues as candidates for point mutationsfor further assessment and refinement of this model as wellas preliminary indications of the requirements for recognitionof this receptor.     

An assessment of the effect of the helix dipole in protein structures

The locations of the cations bound to the peptide group at theC-termini and the anions attached to the main-chain NH groupat the N-termini of helices are analysed. The ions are hardlyfound along the helical axis, where the effect due to the helixmacrodipole is likely to be the maximum. The disposition ofthe ions appears to be controlled more by the stereoelectronicrequirements of the ligand group rather than any long distanceelectric field. This and other related structural observationscall for some circumspection in assigning a role for the helixdipole in protein structure and function.     

The role of electrostatic interactions in the mechanism of peptide bond hydrolysis by a Ser-Lys catalytic dyad

General-base catalysis in the active site of serine proteasesis carried out by the imidazole side chain of a histidine. Duringformation of the transition state, an adjacent carboxylic acidgroup stabilizes the positive charge that forms on the general-basecatalyst and as a result contributes several orders of magnitudeto the catalytic efficiency of these enzymes. In the recentlydiscovered family of self-cleaving proteins exemplified by theLexA repressor of Escherichia coli, instead of the imidazoleof a histidine, the active-site general-base catalyst was foundto be the -amino of a lysine. The considerably higher capacityof the lysine side chain for proton acceptance raises interestingquestions concerning the role of electrostatic interactionsin the mechanism of proton transfer by this highly basic group.The negative charge elimination studies described here and theireffects on the k_max and pK of LexA self-cleavage are consistentwith a model in which electrostatic interactions between anacidic side chain and the general-base catalyst form a barrierto proton transfer. The implications are that the -amino group,unlike the imidazole group, is capable of effecting proton transferwithout the intervention of a countercharge.     

The binding mode of an E-64 analog to the active site of cathepsin B

Two binding modes of the isobutyl-NH-Eps-Leu-Pro inhibitor tocathepsin B have been proposed. Molecular docking using an empiricalforce field was carried out to distinguish between the two modes.The search began with manual docking, followed by random perturbationsof the docking conformation and cycles of Monte Carlo minimization.Finally, molecular dynamics was carried out for the most favorabledocking conformations. The present calculations predict thatthe isobutyl-NH-Eps-Leu-Pro inhibitor preferentially binds tothe S' rather than the S subsites of cathepsin B. The S' bindingmode prediction is supported by the X-ray crystal structureof cathepsin B bound to a closely related ethyl-O-Eps-Ile-Proinhibitor, which was found to bind in the S' subsite with theC-terminal epoxy ring carbon making a covalent bond to the sulfuratom of Cys29. This agreement, in turn, validates our dockingstrategy. Furthermore, the calculations provide evidence thatthe dominant contribution to the total stabilization energyof the enzyme–inhibitor complex stems from the strongelectrostatic interaction between the negatively charged C-terminalcarboxylate group of the ligand and the positively charged imidazoliumrings of His110 and His111. The latter are stabilized and heldin an optimal orientation for interactions with the C-terminalend of the ligand through a salt bridge between the side chainsof His110 and Asp22. By comparison with the crystal structure,some insight into the specificity of the epoxyldipeptide familytowards cathepsin B inhibition has been extracted. Both thecharacteristics of the enzyme (e.g. subsite size and hydrophobicity)as well as the nature of the inhibitor influence the selectivityof an inhibitor towards an enzyme.     

Molecular dynamics of the 5-HT1a receptor and ligands

A 3-D model of the human 5-HT_1a receptor was constructed fromits amino acid sequence by computer graphics techniques, molecularmechanics calculations and molecular dynamics simulations. Themodel has seven -helical membrane spanning segments, which forma central core containing a putative ligand binding site. Electrostaticpotentials 1.4 Å outside the water accessible surfacewere mainly negative on the synaptic side of the receptor modeland at the postulated ligand binding site, and positive in thecytoplasmic domains. The negative electrostatic potentials aroundthe synaptic domains indicate that positively charged ligandsare attracted to the receptor by electrostatic forces. Moleculardynamics simulations of the receptor model with serotonin, ipsapirone,R(–)-methiothepin or S(+)- methiothepin in the centralcore suggested that up to 22 different amino acid residues mayform a ligand binding pocket, and contribute to the specificityof ligand recognition and binding.     

Alpha-Bulges in G Protein-Coupled Receptors

Agonist binding is related to a series of motions in G protein-coupled receptors (GPCRs) that result in the separation of transmembrane helices III and VI at their cytosolic ends and subsequent G protein binding. A large number of smaller motions also seem to be associated with activation. Most helices in GPCRs are highly irregular and often contain kinks, with extensive literature already available about the role of prolines in kink formation and the precise function of these kinks. GPCR transmembrane helices also contain many α-bulges. In this article we aim to draw attention to the role of these α-bulges in ligand and G-protein binding, as well as their role in several aspects of the mobility associated with GPCR activation. This mobility includes regularization and translation of helix III in the extracellular direction, a rotation of the entire helix VI, an inward movement of the helices near the extracellular side, and a concerted motion of the cytosolic ends of the helices that makes their orientation appear more circular and that opens up space for the G protein to bind. In several cases, α-bulges either appear or disappear as part of the activation process.     

Comparison of three algorithms for the assignment of secondary structure in proteins: the advantages of a consensus assignment

Accurate assignments of secondary structures in proteins arecrucial for a useful comparison with theoretical predictions.Three major programs which automatically determine the locationof helices and strands are used for this purpose, namely DSSP,P-Curve and Define. Their results have been compared for a non-redundantdatabase of 154 proteins. On a residue per residue basis, thepercentage match score is only 63% between the three methods.While these methods agree on the overall number of residuesin each of the three states (helix, strand or coil), they differon the number of helices or strands, thus implying a wide discrepancyin the length of assigned structural elements. Moreover, thelength distribution of helices and strands points to the existenceof artefacts inherent to each assignment algorithm. To overcomethese difficulties a consensus assignment is proposed whereeach residue is assigned to the state determined by at leasttwo of the three methods. With this assignment the artefactsof each algorithm are attenuated. The residues assigned in thesame state by the three methods are better predicted than theothers. This assignment will thus be useful for analysing thesuccess rate of prediction methods more accurately.     

Geometry of interaction of metal ions with histidine residues in protein structures

An analysis of the geometry and the orientation of metal ionsbound to histidine residues in proteins is presented. Cationsare found to lie in the imidazole plane along the lone pairon the nitrogen atom. Out of the two tautomeric forms of theimidazole ring, the NE2-protonated form is normally preferred.However, when bound to a metal ion the ND1-protonated form ispredominant and NE2 is the ligand atom. When the metal coordinationis through ND1, steric interactions shift the side chain torsionalangle, X₂ from its preferred value of 90 or 270. The orientationof histidine residues is usually stabilized through hydrogenbonding; ND1-protonated form of a helical residue can form ahydrogen bond with the carbonyl oxygen atom in the precedingturn of the helix. A considerable number of ligands are foundin helices and ß-sheets. A helical residue hound toa heme group is usually found near the C-terminus of the helix.Two ligand groups four residues apart in a helix, or two residuesapart in a ß-strand are used in many proteins to bindmetal ions.     

Interaction of metal ions with carboxylic and carboxamide groups in protein structures

An analysis of the geometry of metal binding by carboxylic andcarboxamide groups in proteins is presented. Most of the ligandsare from aspartic and glutamic acid side chains. Water moleculesbound to carboxylate anions are known to interact with oxygenlone-pairs. However, metal ions are also found to approach thecarboxylate group along the C - O direction. More metal ionsare found to be along the syn than the anti lone-pair direction.This seems to be the result of the stability of the five-memberedring that is formed by the carboxylate anion hydrogen bondedto a ligand water molecule and the metal ion in the syn position.Ligand residues are usually from the helix, turn or regionswith no regular secondary structure. Because of the steric interactionsassociated with bringing all the ligands around a metal center,a calcium ion can bind only near the ends of a helix; a metal,like zinc, with a low coordination number, can bind anywherein the helix. Based on the analysis of the positions of watermolecules in the metal coordination sphere, the sequence ofthe EF hand (a calcium-binding structure) is discussed.     

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

The intestinal guanylyl cyclase-C (GC-C) was originally identifiedas an Escherichia coli heat-stable enterotoxin (STa) receptor.STa stimulates GC-C to much higher activity than the endogenousligands guanylin and uroguanylin, causing severe diarrhea. Toinvestigate the interactions of the endogenous and bacterialligands with GC-C, we designed and characterized a soluble andproperly folded fragment of the extracellular ligand-bindingdomain of GC-C. The membrane-bound guanylyl cyclases exhibita single transmembrane spanning helix and a globularly foldedextracellular ligand-binding domain that comprises about 410of 1050 residues. Based on the crystal structure of the dimerized-bindingdomain of the guanylyl cyclase-coupled atrial natriuretic peptidereceptor and a secondary structure-guided sequence alignment,we generated a model of the extracellular domain of GC-C comprisedof two subdomains. Mapping of mutational and cross-link dataonto this structural model restricts the ligand-binding regionto the membrane proximal subdomain. We thus designed miniGC-C,a 197 amino acid fragment that mimics the ligand-binding membraneproximal subdomain. Cloning, expression and spectroscopic studiesreveal miniGC-C to be a soluble and properly folded proteinwith a distinct secondary and tertiary structure. MiniGC-C bindsSTa with nanomolar affinity.     

Introduction of internal cysteines as conformational probes in yeast phosphoglycerate kinase

Several mutants of yeast phosphoglycerate kinase, each containingonly one internal cysteine residue, were constructed from asingle mutant devoid of cysteine. These cysteines were introducedas local conformational probes in selected buried positions.The enzyme activity, conformational characteristics and stabilityindicated that the mutations introduced only small perturbationsin the molecule. The folding–unfolding process mediatedby guanidine hydrochloride under equilibrium conditions wasstudied by following the variations in ellipticity and the reactivityof the cysteine residue towards 5,5'-dithiobis(nitrobenzoate).The process was found to be reversible except for mutant C97A,V49C,suggesting that this region located in helix I might be crucialin determining an intermediate on the folding pathway. The transitionsobtained by the two signals did not coincide, indicating thatthe local structures, in several parts inside the molecule,are more sensitive to the denaturant than the overall conformation.     

Computational analysis of alpha-helical membrane protein structure: implications for the prediction of 3D structural models

Relatively little has been known about the structure of alpha-helical membrane proteins, since until recently few structures had been crystallized. These limited data have restricted structural analyses to the prediction of secondary structure, rather than tertiary folds. In order to address this, this paper describes an analysis of the 23 available membrane protein structures. A number of findings are made that are of particular relevance to transmembrane helix packing: (1) on average lipid-tail-accessible transmembrane residues are significantly more hydrophobic, less conserved and contain different residue types to buried residues; (2) charged residues are not always buried and, when accessible to membrane lipid tails, few are paired with another charge and instead they often interact with phospholipid head-groups or with other residue types; (3) a significant proportion of lipid-tail-accessible charged and polar residues form hydrogen bonds only with residues one turn away in the same helix (intra-helix); (4) pore-lining residues are usually hydrophobic and it is difficult to distinguish them from buried residues in terms of either residue type or conservation; and (5) information was gained about the proportion of helices that tend to contribute to lining a pore and the resulting pore diameter. These findings are discussed with relevance to the prediction of membrane protein 3D structure.     

The prediction and orientation of {alpha}-helices from sequence alignments: the combined use of environment-dependent substitution tables, Fourier transform methods and helix capping rules

Amino acid substitution tables are used to estimate the extentto which amino acids in families of homologous proteins areexposed to the solvent. The approach depends on the comparisonof difference environment-dependent tables for solvent accessible/inaccessibleresidues with amino acid substitutions at each position in analigned set of sequences. The periodicity in the predicted accessible/inaccessibleresidues is calculated using a Fourier transform procedure modifiedfrom that used to calculate hydrophobic moments. a-Helices areidentified from the characteristic periodicities and the solventaccessible face of the helix is defined. The initial helix predictionsare refined using rules for identifying the N- and C-terminiof helices from sequence alignments. These rules have been definedfrom a study of protein structures. The combined method correctlypredicts 79% of the residues in helices and incorrectly predictsonly 12% of the nonhelical residues as helical. In addition,since the method is reliable at predicting the correct numberof helices in the correct position in the sequence and sinceit also predicts the internal face of each helix, the resultscan be used to postulate 3-D arrangements of the secondary structureelements.