to their ubiquitous expression and potential as drug targets. Molecular interactions between liga... more to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. This probe, 125 I-desaminotyrosyl-Gly-[Nle 28,31 ,pNO 2 -Phe 33 ]CCK-(26 -33), binds specifically, saturably, and with high affinity (K i ؍ 3.3 nM) and has full agonist activity. This makes likely its being sited in a natural position within the receptor. As substrate, we used CHO-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor. Covalent labeling of the appropriate M r ؍ 85,000 -95,000 plasma membrane glycoprotein with core of M r ؍ 42,000 was established by SDS-polyacrylamide gel electrophoresis and autoradiography. A single domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure liquid chromatography. The site of interaction was further defined by additional proteolysis with trypsin, with purification of the labeled fragment, followed by manual Edman degradation and radiochemical sequencing. This demonstrated that Trp 39 was specifically labeled and likely resides proximate to the carboxyl-terminal pNO 2 -Phe 33 residue of the probe. A model of this ligandbound receptor has been constructed and will be used to plan future experiments to refine our understanding of this interaction.
The receptors for cholecystokinin (CCK) peptides are guanine nucleotide-binding protein-coupled r... more The receptors for cholecystokinin (CCK) peptides are guanine nucleotide-binding protein-coupled receptors in the rhodopsin/b-adrenergic receptor family. The molecular basis of natural ligand binding to the type A CCK receptor has been studied using ligand structure-activity series, receptor mutagenesis, and photoaffinity labeling studies. These have focused attention on the extracellular loop and tail domains, with the most direct insights coming from intrinsic photoaffinity labeling studies. A model of the binding of CCK to this receptor is consistent with all these studies. This model places the carboxyl terminus of CCK adjacent to the amino-terminal tail outside of transmembrane segment 1, the midregion of the peptide adjacent to the third extracellular loop outside of transmembrane segment 7, and includes a chargecharge interaction between peptide residue tyrosine-sulfate 27 and the arginine residue in the second extracellular loop of the receptor in position 197.
MD Display was developed as a means of visuali~.ing molecular dynamic trajectories generated by A... more MD Display was developed as a means of visuali~.ing molecular dynamic trajectories generated by Amber. i The program runs on Silicon Graphics workstations, and features a simple user interface, and convenient display and analysis options. The program has now been extended to accept input from several other molecular dynamics programs.
The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-co... more The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D 2 (PGD 2) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD 2 binding affinity, suggesting that these residues play a role in binding PGD 2. In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD 2 , whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE 2 by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD 2 binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.
An understanding of the molecular basis of drug action provides opportunities for refinement of d... more An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of attachment for the photolabile moiety of the antagonist probe deep within the receptor's membrane-spanning region at Leu 88 , a residue within transmembrane segment two. In contrast, the agonist probe labeled a region including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular models for the antagonist-receptor complex. These models provided a means for visualization of physically plausible ligand-receptor interactions in the context of all currently available biological data that address small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces powerful tools and a strategy for direct and selective investigation of non-peptidyl ligand binding to peptide receptors.
Journal of the American Chemical Society, May 1, 1990
Molecular dynamic simulations are used to predict the binding affinity in host-guest systems by t... more Molecular dynamic simulations are used to predict the binding affinity in host-guest systems by the thermodynamic cycleperturbation method. The relative free energy of solvation of Na+ and K+ in methanol (19.8 and 19.1 kcal mol-') and the relative free energy of binding of Na+ and K+ to 18-crown-6 in methanol (-3.8 and-3.0 kcal mol-') are calculated by thermodynamic integration and thermodynamic perturbation, respectively. These results are in reasonable agreement with the experimental values, 17.3 and-2.47 kcal mol-', respectively. In addition, the contributions to the relative free energy from the internal energy and entropy are calculated. Finally, a detailed analysis is made of the structure and its fluctuations in this system to provide additional insight to the selectivity of binding. Since their discovery in 1967 by Pedersen' at Du Pont, crown ethers have been a very important member of the class of molecules known as organic Although synthetic, crown ethers share with enzymes and other biological molecules the capacity of recognizing and selectively binding ions or other molecules. Crown ethers display a wide range of binding specificities, in part because they range in size from a ring containing as few as 9 ring atoms to more than 30.4 In addition, the ring can be modified to decrease its flexibility by replacing the saturated carbon-carbon linking group with aromatic carbon^.^ Substitutions can also be made in which chains are added in such a fashion that they aid in complex formation from above or below the plane of the ring.* Originally, it was proposed that the selectivity of crown ethers depended mainly on the size of the guest relative to the cavity
Distinct spatial approximations between residues within the secretin pharmacophore and its recept... more Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile pbenzoyl-L-phenylalanine into the position of His 1 of rat secretin ([Bpa 1 ,Tyr 10 ]secretin-27). Because His 1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions ؊1 (rat [Bpa ؊1 ,Tyr 10 ]secretin-27) and ؊2 (rat [Bpa ؊2 ,Gly ؊1 ,-Tyr 10 ]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptorbearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position ؊2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.
A thermodynamic perturbation method, using a Monte Carlo procedure, has been applied to the calcu... more A thermodynamic perturbation method, using a Monte Carlo procedure, has been applied to the calculation of the free energy differences between ion–water clusters containing a given number of water molecules (n=1,...,6) and different monovalent ions, i.e., Li+, Na+, K+, F−, and Cl−. To our knowledge, this is the first application of the free energy perturbation approach with a nonadditive intermolecular potential. The results confirm the usefulness of the method and yield free energy differences of hydration which are in generally very good agreement with experiment. Our calculations have also revealed some structural changes which take place within the first hydration shell when one ion is replaced by another in the hexahydrate clusters. For Li+ one obtains a well defined 4+2 mean structure, whereas for a larger ion such as K+ the solvation structure is closer to a 5+1 type. For anions, this tendency is reversed, i.e., for F− one finds a well defined four coordinate structure for a cluster with four water molecules, but for the larger anion Cl− the cluser tends to be a 3+1 structure. In addition, favorable water–water interactions are apparent in anion–water clusters, but not in cation–water clusters.
A parallel version of the popular molecular mechanics package AMBER suitable for execution on wor... more A parallel version of the popular molecular mechanics package AMBER suitable for execution on workstation clusters has been developed. Computer-intensive portions of molecular dynamics or free-energy perturbation computations, such as nonbonded pair list generation or calculation of nonbonded energies and forces, are distributed across a collection of Unix workstations linked by Ethernet or FDDI connections. This parallel implementation utilizes the message-passing software PVM (Parallel Virtual Machine) from Oak Ridge National Laboratory to coordinate data exchange and processor synchronization. Test simulations performed for solvated peptide, protein, and lipid bilayer systems indicate that reasonable parallel efficiency (70-90%) and computational speedup (2-5 X serial computer runtimes) can be achieved with small workstation clusters (typically six to eight machines) for typical biomolecular simulation problems. PVM-AMBER is also easily and rapidly portable to different hardware platforms due to the availability of PVM for numerous computers. The current version of PVM-AMBER has been tested successfully on Silicon Graphics, IBM RS6000, DEC ALPHA, and HP 735 workstation clusters and heterogeneous clusters of these machines, as well as on CRAY T3D and Kendall Square KSR2 parallel supercomputers. Thus, PVM-AMBER provides a simple and cost-effective mechanism for parallel molecular dynamics simulations on readily available hardware platforms. Factors that affect the efficiency of this approach are discussed. 0 1995 by John Wiley & Sons, Inc. systems. Techniques such as Monte Carlo or molecular dynamics statistical mechanical calculations are used routinely to study condensed-phase systems as well as polymers and large biomolecules. These simulation tools can provide information and insight that is difficult to obtain from direct experimental measurements. However,
Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domai... more Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg E from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg E domain was created using the homodimeric, fourhelix bundle structure of the Tar S domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg E periplasmic domain is very similar to the Tar S domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.
We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in b... more We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule, but all neighboring side chain positions are preserved and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen-bonding network in the biotin binding pocket. High resolution crystal structure information is crucial for the prediction and design of proteinprotein and protein-ligand interactions, and has been particularly useful in explaining how point mutations or ligand modifications impact ligand recognition and binding affinity. However, there are a growing number of examples of point mutations, often quite far from the ligand binding site, that affect ligand binding affinities even when no structural changes are observed.
The amino terminus of the secretin receptor (SecR) is known to be critical for natural agonist ac... more The amino terminus of the secretin receptor (SecR) is known to be critical for natural agonist action, although the role it plays is still unclear. We have demonstrated that photolabile residues within both the amino-terminal (position 6) and carboxylterminal (positions 22 and 26) halves of secretin each covalently label receptor amino-terminal tail residues [
to their ubiquitous expression and potential as drug targets. Molecular interactions between liga... more to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. This probe, 125 I-desaminotyrosyl-Gly-[Nle 28,31 ,pNO 2 -Phe 33 ]CCK-(26 -33), binds specifically, saturably, and with high affinity (K i ؍ 3.3 nM) and has full agonist activity. This makes likely its being sited in a natural position within the receptor. As substrate, we used CHO-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor. Covalent labeling of the appropriate M r ؍ 85,000 -95,000 plasma membrane glycoprotein with core of M r ؍ 42,000 was established by SDS-polyacrylamide gel electrophoresis and autoradiography. A single domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure liquid chromatography. The site of interaction was further defined by additional proteolysis with trypsin, with purification of the labeled fragment, followed by manual Edman degradation and radiochemical sequencing. This demonstrated that Trp 39 was specifically labeled and likely resides proximate to the carboxyl-terminal pNO 2 -Phe 33 residue of the probe. A model of this ligandbound receptor has been constructed and will be used to plan future experiments to refine our understanding of this interaction.
The receptors for cholecystokinin (CCK) peptides are guanine nucleotide-binding protein-coupled r... more The receptors for cholecystokinin (CCK) peptides are guanine nucleotide-binding protein-coupled receptors in the rhodopsin/b-adrenergic receptor family. The molecular basis of natural ligand binding to the type A CCK receptor has been studied using ligand structure-activity series, receptor mutagenesis, and photoaffinity labeling studies. These have focused attention on the extracellular loop and tail domains, with the most direct insights coming from intrinsic photoaffinity labeling studies. A model of the binding of CCK to this receptor is consistent with all these studies. This model places the carboxyl terminus of CCK adjacent to the amino-terminal tail outside of transmembrane segment 1, the midregion of the peptide adjacent to the third extracellular loop outside of transmembrane segment 7, and includes a chargecharge interaction between peptide residue tyrosine-sulfate 27 and the arginine residue in the second extracellular loop of the receptor in position 197.
MD Display was developed as a means of visuali~.ing molecular dynamic trajectories generated by A... more MD Display was developed as a means of visuali~.ing molecular dynamic trajectories generated by Amber. i The program runs on Silicon Graphics workstations, and features a simple user interface, and convenient display and analysis options. The program has now been extended to accept input from several other molecular dynamics programs.
The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-co... more The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D 2 (PGD 2) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD 2 binding affinity, suggesting that these residues play a role in binding PGD 2. In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD 2 , whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE 2 by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD 2 binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.
An understanding of the molecular basis of drug action provides opportunities for refinement of d... more An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of attachment for the photolabile moiety of the antagonist probe deep within the receptor's membrane-spanning region at Leu 88 , a residue within transmembrane segment two. In contrast, the agonist probe labeled a region including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular models for the antagonist-receptor complex. These models provided a means for visualization of physically plausible ligand-receptor interactions in the context of all currently available biological data that address small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces powerful tools and a strategy for direct and selective investigation of non-peptidyl ligand binding to peptide receptors.
Journal of the American Chemical Society, May 1, 1990
Molecular dynamic simulations are used to predict the binding affinity in host-guest systems by t... more Molecular dynamic simulations are used to predict the binding affinity in host-guest systems by the thermodynamic cycleperturbation method. The relative free energy of solvation of Na+ and K+ in methanol (19.8 and 19.1 kcal mol-') and the relative free energy of binding of Na+ and K+ to 18-crown-6 in methanol (-3.8 and-3.0 kcal mol-') are calculated by thermodynamic integration and thermodynamic perturbation, respectively. These results are in reasonable agreement with the experimental values, 17.3 and-2.47 kcal mol-', respectively. In addition, the contributions to the relative free energy from the internal energy and entropy are calculated. Finally, a detailed analysis is made of the structure and its fluctuations in this system to provide additional insight to the selectivity of binding. Since their discovery in 1967 by Pedersen' at Du Pont, crown ethers have been a very important member of the class of molecules known as organic Although synthetic, crown ethers share with enzymes and other biological molecules the capacity of recognizing and selectively binding ions or other molecules. Crown ethers display a wide range of binding specificities, in part because they range in size from a ring containing as few as 9 ring atoms to more than 30.4 In addition, the ring can be modified to decrease its flexibility by replacing the saturated carbon-carbon linking group with aromatic carbon^.^ Substitutions can also be made in which chains are added in such a fashion that they aid in complex formation from above or below the plane of the ring.* Originally, it was proposed that the selectivity of crown ethers depended mainly on the size of the guest relative to the cavity
Distinct spatial approximations between residues within the secretin pharmacophore and its recept... more Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile pbenzoyl-L-phenylalanine into the position of His 1 of rat secretin ([Bpa 1 ,Tyr 10 ]secretin-27). Because His 1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions ؊1 (rat [Bpa ؊1 ,Tyr 10 ]secretin-27) and ؊2 (rat [Bpa ؊2 ,Gly ؊1 ,-Tyr 10 ]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptorbearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position ؊2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.
A thermodynamic perturbation method, using a Monte Carlo procedure, has been applied to the calcu... more A thermodynamic perturbation method, using a Monte Carlo procedure, has been applied to the calculation of the free energy differences between ion–water clusters containing a given number of water molecules (n=1,...,6) and different monovalent ions, i.e., Li+, Na+, K+, F−, and Cl−. To our knowledge, this is the first application of the free energy perturbation approach with a nonadditive intermolecular potential. The results confirm the usefulness of the method and yield free energy differences of hydration which are in generally very good agreement with experiment. Our calculations have also revealed some structural changes which take place within the first hydration shell when one ion is replaced by another in the hexahydrate clusters. For Li+ one obtains a well defined 4+2 mean structure, whereas for a larger ion such as K+ the solvation structure is closer to a 5+1 type. For anions, this tendency is reversed, i.e., for F− one finds a well defined four coordinate structure for a cluster with four water molecules, but for the larger anion Cl− the cluser tends to be a 3+1 structure. In addition, favorable water–water interactions are apparent in anion–water clusters, but not in cation–water clusters.
A parallel version of the popular molecular mechanics package AMBER suitable for execution on wor... more A parallel version of the popular molecular mechanics package AMBER suitable for execution on workstation clusters has been developed. Computer-intensive portions of molecular dynamics or free-energy perturbation computations, such as nonbonded pair list generation or calculation of nonbonded energies and forces, are distributed across a collection of Unix workstations linked by Ethernet or FDDI connections. This parallel implementation utilizes the message-passing software PVM (Parallel Virtual Machine) from Oak Ridge National Laboratory to coordinate data exchange and processor synchronization. Test simulations performed for solvated peptide, protein, and lipid bilayer systems indicate that reasonable parallel efficiency (70-90%) and computational speedup (2-5 X serial computer runtimes) can be achieved with small workstation clusters (typically six to eight machines) for typical biomolecular simulation problems. PVM-AMBER is also easily and rapidly portable to different hardware platforms due to the availability of PVM for numerous computers. The current version of PVM-AMBER has been tested successfully on Silicon Graphics, IBM RS6000, DEC ALPHA, and HP 735 workstation clusters and heterogeneous clusters of these machines, as well as on CRAY T3D and Kendall Square KSR2 parallel supercomputers. Thus, PVM-AMBER provides a simple and cost-effective mechanism for parallel molecular dynamics simulations on readily available hardware platforms. Factors that affect the efficiency of this approach are discussed. 0 1995 by John Wiley & Sons, Inc. systems. Techniques such as Monte Carlo or molecular dynamics statistical mechanical calculations are used routinely to study condensed-phase systems as well as polymers and large biomolecules. These simulation tools can provide information and insight that is difficult to obtain from direct experimental measurements. However,
Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domai... more Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg E from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg E domain was created using the homodimeric, fourhelix bundle structure of the Tar S domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg E periplasmic domain is very similar to the Tar S domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.
We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in b... more We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule, but all neighboring side chain positions are preserved and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen-bonding network in the biotin binding pocket. High resolution crystal structure information is crucial for the prediction and design of proteinprotein and protein-ligand interactions, and has been particularly useful in explaining how point mutations or ligand modifications impact ligand recognition and binding affinity. However, there are a growing number of examples of point mutations, often quite far from the ligand binding site, that affect ligand binding affinities even when no structural changes are observed.
The amino terminus of the secretin receptor (SecR) is known to be critical for natural agonist ac... more The amino terminus of the secretin receptor (SecR) is known to be critical for natural agonist action, although the role it plays is still unclear. We have demonstrated that photolabile residues within both the amino-terminal (position 6) and carboxylterminal (positions 22 and 26) halves of secretin each covalently label receptor amino-terminal tail residues [
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Papers by Terry Lybrand