Page 1. Journal of Protein Chemistry, Vol. 5, No. 1, 1986 Comparisons of Creatine Kinase Primary ... more Page 1. Journal of Protein Chemistry, Vol. 5, No. 1, 1986 Comparisons of Creatine Kinase Primary Structures Patricia C. Babbitt, ~ George L. Kenyon, 1'1° Irwin D. Kuntz, 1 Fred E. Cohen, 1 John D. Baxter, 2 Pamela A. Benfield ...
The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an aut... more The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg66...
Proceedings of the National Academy of Sciences, 2005
Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have... more Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoform-specific effects on the deposition or clearance of amyloid β (Aβ) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on Aβ production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased Aβ production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in Aβ production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptor-related protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which ab...
Tryptases I and II were heterologously expressed and purified in yeast to functionally characte... more Tryptases I and II were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. I and II tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for -tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96, P3 Asp-60B and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of -tryptases show consistency with the specificity data presented here.
The ability to generate feasible binding orientations of a small molecule within a site of known ... more The ability to generate feasible binding orientations of a small molecule within a site of known structure is important for ligand design. We present a method that combines a rapid, geometric docking algorithm with the evaluation of molecular mechanics interaction energies. The computational costs of evaluation are minimal because we precalculate the receptor-dependent terms in the potential function at points on a threedimensional grid. In four test cases where the components of crystallographically determined complexes are redocked, the "force field' score correctly identifies the family of orientations closest to the experimental binding geometry. Scoring functions that consider only steric factors or only electrostatic factors are less successful. The force field function will play an important role in our efforts to search databases for potential lead compounds.
To introduce selection into a model of coalescence, I explore the use of modified integer partiti... more To introduce selection into a model of coalescence, I explore the use of modified integer partitions that allow the identification of a preferred lineage. I show that a partition-partition transition matrix, along with Monte Carlo discrete time kinetics, treats both the neutral case and a wide range of positive and negative selection pressures for small population sizes. Selection pressure causes multiple collisions per generation, short coalescence times, increased lengths of terminal branches, increased tree asymmetry, and dependence of coalescence times on the logarithm of population size. These features are consistent with higher order coalescences that permit multiple collisions per generation. While the treatment is exact in terms of the simplified Wright-Fisher model used, it is not easily extended to large population size. Keywords: Selection, Coalescence, Integer Partitions, Multiple Collisions, Tree Asymmetry.
ABSTRACT The ability to propose reasonable ligand-receptor binding geometries is crucial to the s... more ABSTRACT The ability to propose reasonable ligand-receptor binding geometries is crucial to the success of structure-based drug design. One approach is to “dock” molecules together in many ways and then “score” or evaluate each orientation; in a database of compounds, those which score well should be more likely to bind to the target macromolecule. A method that combines a rapid, geometric docking algorithm with the evaluation of molecular mechanics interaction energies is presented. The “force field score” is successful in identifying the experimental binding mode in four systems, and represents an improvement over the other scoring methods tested. The degree of orientational sampling required to reproduce and identify the known geometries, with and without energy-minimization, is also investigated. Both scoring and sampling issues are of paramount importance to the usefulness of molecular docking in real-life applications.
... Elaine Meng recehred her BS degree in pharmacy at the University of Cincinnati in 1988 and he... more ... Elaine Meng recehred her BS degree in pharmacy at the University of Cincinnati in 1988 and her Ph.D. degree in ... Atomic coordinates for receptor macromolecules can be obtained through X-ray crystallography, nuclear magnetic resonance (NMR), and homology modeling. ...
determine the solution structure of a 141 residue protein containing the GTPase activating domain... more determine the solution structure of a 141 residue protein containing the GTPase activating domain from the alpha chain of the heterotrimeric G University of California at protein G s. The domain contains six a-helices and is stable and structured San Francisco, San Francisco in solution despite having been excised from the intact G s protein. The CA 94143-0446, USA N-terminal ten and C-terminal 11 residues of the protein are unstructured 2 Department of Pharmacology in solution while the core is well determined by the 2483 distance and University of California at torsion restraints derived from the NMR spectra. The final ensemble of 14 San Francisco, San Francisco structures, generated with a hybrid distance geometry/simulated CA 94143-0446, USA annealing protocol, have an average to-the-mean backbone root-meansquare deviation of 0.39 Å for the core residues 89 to 201. The majority of the structure is remarkably similar to that observed for the cognate domains in crystal structures of the homologous proteins a t and a i1. However, the orientations of the second helix and the subsequent interhelical loops differ markedly among the three proteins. This structural divergence, along with functional studies of chimeric proteins, suggests that this region of the domain interacts with either the downstream effector adenylyl cyclase or with some other intermediary protein.
A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal caviti... more A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal cavities is examined to study the effect of protein motions on ligand motion and internal cavity fluctuations. Average structural and energetic properties indicate that the simulation is well behaved. The average protein volume is similar to the volume of the X-ray model and the main-chain atom root-mean-square deviation between the X-ray model and the average dynamical structure is 1.25 A. The protein volume oscillates 3 to 43;) around the volume of the X-ray structure. These fluctuations lead to changes in the internal free volume and in the size, shape and location of atom-sized cavity features. Transient cavities produced in the simulation have a crucial role in the movement of two of the ligands. One of the ligands escapes to the protein surface. whilst a second ligand travels through the protein interior. Complex gating processes involving several protein residues are responsible for producing the necessary pores through which the ligand passes bet,ween transient cavities or packing defects.
For the first time a general shape-search docking algorithm (DOCK) has been applied to the minor ... more For the first time a general shape-search docking algorithm (DOCK) has been applied to the minor and major grooves of A-, B- and Z-type DNA dodecamers and to an intercalation site in a B-DNA-type hexamer. Both experimentally and theoretically derived geometries for the various DNA fragments were used. The DOCK searches were carried out on a subset of the Cambridge Crystallographic Database, consisting of almost 10,000 molecules. One of the molecules that scored best in terms of the DOCK algorithm was CC-1065, a potent antitumor agent known to (covalently) bind the AT-rich parts of the minor groove of B-DNA. Several known DNA-binding agents also scored highly. Molecules with shapes complementary to A-, B- and Z-type DNA were indicated by DOCK. In addition, compounds were extracted from the database that might be selective for the GC-rich regions of the minor groove of B-DNA. Many of the compounds in the present study may serve as a starting point for further molecular design of novel DNA-binding ligands.
... (14) L. E. Orgel and R. S. Mulliken, J. Am. Chem. Soc., 79, 4839 (1957). (15) A. A. Sandoval ... more ... (14) L. E. Orgel and R. S. Mulliken, J. Am. Chem. Soc., 79, 4839 (1957). (15) A. A. Sandoval and MW Hanna, J. Phys. Chem., 70, 1203 (1966). (16) J. Crossley and C. P. Smyth, J.Am. Chem. Soc., 91, 2482 (1969). (17) M. D. Magee and S. Walker, J. Chem. ...
Page 1. Journal of Protein Chemistry, Vol. 5, No. 1, 1986 Comparisons of Creatine Kinase Primary ... more Page 1. Journal of Protein Chemistry, Vol. 5, No. 1, 1986 Comparisons of Creatine Kinase Primary Structures Patricia C. Babbitt, ~ George L. Kenyon, 1'1° Irwin D. Kuntz, 1 Fred E. Cohen, 1 John D. Baxter, 2 Pamela A. Benfield ...
The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an aut... more The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg66...
Proceedings of the National Academy of Sciences, 2005
Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have... more Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoform-specific effects on the deposition or clearance of amyloid β (Aβ) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on Aβ production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased Aβ production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in Aβ production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptor-related protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which ab...
Tryptases I and II were heterologously expressed and purified in yeast to functionally characte... more Tryptases I and II were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme. Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases. Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine. I and II tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine. Measurement of kinetic constants with multiple substrates designed for -tryptases reveal that selectivity is highly dependent on ground state substrate binding. Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified. Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96, P3 Asp-60B and Glu-217, and P1 Asp-189. Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor. Finally, the cleavage sites of several physiologically relevant substrates of -tryptases show consistency with the specificity data presented here.
The ability to generate feasible binding orientations of a small molecule within a site of known ... more The ability to generate feasible binding orientations of a small molecule within a site of known structure is important for ligand design. We present a method that combines a rapid, geometric docking algorithm with the evaluation of molecular mechanics interaction energies. The computational costs of evaluation are minimal because we precalculate the receptor-dependent terms in the potential function at points on a threedimensional grid. In four test cases where the components of crystallographically determined complexes are redocked, the "force field' score correctly identifies the family of orientations closest to the experimental binding geometry. Scoring functions that consider only steric factors or only electrostatic factors are less successful. The force field function will play an important role in our efforts to search databases for potential lead compounds.
To introduce selection into a model of coalescence, I explore the use of modified integer partiti... more To introduce selection into a model of coalescence, I explore the use of modified integer partitions that allow the identification of a preferred lineage. I show that a partition-partition transition matrix, along with Monte Carlo discrete time kinetics, treats both the neutral case and a wide range of positive and negative selection pressures for small population sizes. Selection pressure causes multiple collisions per generation, short coalescence times, increased lengths of terminal branches, increased tree asymmetry, and dependence of coalescence times on the logarithm of population size. These features are consistent with higher order coalescences that permit multiple collisions per generation. While the treatment is exact in terms of the simplified Wright-Fisher model used, it is not easily extended to large population size. Keywords: Selection, Coalescence, Integer Partitions, Multiple Collisions, Tree Asymmetry.
ABSTRACT The ability to propose reasonable ligand-receptor binding geometries is crucial to the s... more ABSTRACT The ability to propose reasonable ligand-receptor binding geometries is crucial to the success of structure-based drug design. One approach is to “dock” molecules together in many ways and then “score” or evaluate each orientation; in a database of compounds, those which score well should be more likely to bind to the target macromolecule. A method that combines a rapid, geometric docking algorithm with the evaluation of molecular mechanics interaction energies is presented. The “force field score” is successful in identifying the experimental binding mode in four systems, and represents an improvement over the other scoring methods tested. The degree of orientational sampling required to reproduce and identify the known geometries, with and without energy-minimization, is also investigated. Both scoring and sampling issues are of paramount importance to the usefulness of molecular docking in real-life applications.
... Elaine Meng recehred her BS degree in pharmacy at the University of Cincinnati in 1988 and he... more ... Elaine Meng recehred her BS degree in pharmacy at the University of Cincinnati in 1988 and her Ph.D. degree in ... Atomic coordinates for receptor macromolecules can be obtained through X-ray crystallography, nuclear magnetic resonance (NMR), and homology modeling. ...
determine the solution structure of a 141 residue protein containing the GTPase activating domain... more determine the solution structure of a 141 residue protein containing the GTPase activating domain from the alpha chain of the heterotrimeric G University of California at protein G s. The domain contains six a-helices and is stable and structured San Francisco, San Francisco in solution despite having been excised from the intact G s protein. The CA 94143-0446, USA N-terminal ten and C-terminal 11 residues of the protein are unstructured 2 Department of Pharmacology in solution while the core is well determined by the 2483 distance and University of California at torsion restraints derived from the NMR spectra. The final ensemble of 14 San Francisco, San Francisco structures, generated with a hybrid distance geometry/simulated CA 94143-0446, USA annealing protocol, have an average to-the-mean backbone root-meansquare deviation of 0.39 Å for the core residues 89 to 201. The majority of the structure is remarkably similar to that observed for the cognate domains in crystal structures of the homologous proteins a t and a i1. However, the orientations of the second helix and the subsequent interhelical loops differ markedly among the three proteins. This structural divergence, along with functional studies of chimeric proteins, suggests that this region of the domain interacts with either the downstream effector adenylyl cyclase or with some other intermediary protein.
A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal caviti... more A 96 picosecond dynamics trajectory of myoglobin with five xenon-probe ligands in internal cavities is examined to study the effect of protein motions on ligand motion and internal cavity fluctuations. Average structural and energetic properties indicate that the simulation is well behaved. The average protein volume is similar to the volume of the X-ray model and the main-chain atom root-mean-square deviation between the X-ray model and the average dynamical structure is 1.25 A. The protein volume oscillates 3 to 43;) around the volume of the X-ray structure. These fluctuations lead to changes in the internal free volume and in the size, shape and location of atom-sized cavity features. Transient cavities produced in the simulation have a crucial role in the movement of two of the ligands. One of the ligands escapes to the protein surface. whilst a second ligand travels through the protein interior. Complex gating processes involving several protein residues are responsible for producing the necessary pores through which the ligand passes bet,ween transient cavities or packing defects.
For the first time a general shape-search docking algorithm (DOCK) has been applied to the minor ... more For the first time a general shape-search docking algorithm (DOCK) has been applied to the minor and major grooves of A-, B- and Z-type DNA dodecamers and to an intercalation site in a B-DNA-type hexamer. Both experimentally and theoretically derived geometries for the various DNA fragments were used. The DOCK searches were carried out on a subset of the Cambridge Crystallographic Database, consisting of almost 10,000 molecules. One of the molecules that scored best in terms of the DOCK algorithm was CC-1065, a potent antitumor agent known to (covalently) bind the AT-rich parts of the minor groove of B-DNA. Several known DNA-binding agents also scored highly. Molecules with shapes complementary to A-, B- and Z-type DNA were indicated by DOCK. In addition, compounds were extracted from the database that might be selective for the GC-rich regions of the minor groove of B-DNA. Many of the compounds in the present study may serve as a starting point for further molecular design of novel DNA-binding ligands.
... (14) L. E. Orgel and R. S. Mulliken, J. Am. Chem. Soc., 79, 4839 (1957). (15) A. A. Sandoval ... more ... (14) L. E. Orgel and R. S. Mulliken, J. Am. Chem. Soc., 79, 4839 (1957). (15) A. A. Sandoval and MW Hanna, J. Phys. Chem., 70, 1203 (1966). (16) J. Crossley and C. P. Smyth, J.Am. Chem. Soc., 91, 2482 (1969). (17) M. D. Magee and S. Walker, J. Chem. ...
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