Ligand-protein docking and rational drug design.

Over the past year there have been some interesting and significant advances in computer-based ligand-protein docking techniques and related rational drug-design tools, including flexible ligand docking and better estimation of binding free energies and solvation energies. As a result, the successful use of computational tools to help generate interesting new guide (lead) compounds for targeted receptors is becoming more commonplace.

Polymorphic amino acid variations in HLA-DQ are associated with systematic physical property changes and occurrence of IDDM. Members of the Swedish Childhood Diabetes Study.

The association between human leukocyte antigen (HLA) and insulin-dependent diabetes was studied in a large population-based investigation using genotyping of 425 new-onset patients, 0-14 years of age, and 367 matched control subjects. As many as 97% of patients compared with 75% of control subjects were positive for one or several of DQA1*0301, DQA1*0501, DQB1*0302, or DQB1*0201. Asp-57 DQB was present among 28% of patients, indicating that this residue alone does not confer protection. Combining Asp-57 DQB1 with either Arg-52 DQA1 or Leu-69 DQA1 did not explain susceptibility or protection either. DQA1*0301-DQB1*0302 (DQ8) and DQA1*0301-DQB1*0301 (DQ7) are identical except for four amino acid substitutions in the beta-chain, but DQ8 was positively (odds ratio 8.07; P < 0.001) and DQ7 negatively (odds ratio 0.38; P < 0.001) associated with the disease. Molecular modeling was used to determine whether physiochemical properties such as steric factors and surface electrostatic potentials also differ in a systematic way for various DQ molecules. Amino acids were substituted systematically at the four polymorphic sites, and the solvent-accessible surfaces and electrostatic potentials were computed for each molecule. Dramatic alterations in electrostatic potential were seen for double substitutions at position 45 (G45E) and 57 (A57D) of DQB1. The variation of physicochemical properties due to polymorphic substitutions may be significant to the mechanism of HLA-DQ association with insulin-dependent diabetes, via the effect these property variations have on peptide antigen binding selectivity and subsequent interactions with specific T-cell receptors.

Three-dimensional structure for the beta 2 adrenergic receptor protein based on computer modeling studies.

Computer-aided model building techniques have been used to construct three-dimensional model structures for hamster beta 2 adrenergic receptor. Experimental data were used as constraints to guide the model building procedure, and a number of rather strict criteria were applied to assess the physical plausibility of model structures. We present details of our best model structure to date, which is consistent with a large body of experimental data. We also discuss in detail our model building procedures and evaluation criteria, which we believe may be of general utility in modeling projects.

Computer modeling of actinomycin D interactions with double-helical DNA.

We have performed molecular mechanical calculations on intercalation complexes of actinomycin D with a series of base-paired hexanucleoside pentaphosphates; d(GCGCGC)2, d(GCCGGC)2, d(GCATGC)2, d(GCTAGC)2 and d(ATGCAT)2. Our results are in good agreement with previous experimental work on sequence selectivity. The results provide a rationalization for the strong preference of actinomycin D to intercalate on the 3' side of guanine residues, consistent with previously proposed models. Finally, the computed structures for d(ATGCAT)2-actinomycin D complexes have been compared with two-dimensional nuclear magnetic resonance nuclear Overhauser effect experimental results. To our knowledge, this is the first extensive comparison of molecular mechanical model structures for a drug-DNA complex with experimental solution phase data. We find generally good agreement between our computational models and the experimental solution phase structures.

Three-dimensional models for agonist and antagonist complexes with beta 2 adrenergic receptor.

Computer-modeling techniques have been used to generate docked complexes for a series of beta adrenergic agonists and antagonists with a three-dimensional model of the beta 2 adrenergic receptor. For all ligands tested, it proved possible to dock low-energy conformers in the receptor model, with sensible electrostatic, steric, and hydrogen-bonding interactions, many of which are supported by experimental studies of the beta 2 receptor. Our results illustrate the power of molecular modeling techniques, when coupled with appropriate experimental methods and data, to investigate structure-function properties of integral membrane receptor proteins that cannot yet be studied by direct structural methods.

A peptide agonist acts by occupation of a monomeric G protein-coupled receptor: dual sites of covalent attachment to domains near TM1 and TM7 of the same molecule make biologically significant domain-swapped dimerization unlikely.

Membrane receptor dimerization is a well-established event for initiation of signaling at growth factor receptors and has been postulated to exist for G protein-coupled receptors, based on correction of nonfunctional truncated, mutant, or chimeric constructs by coexpression of appropriate normal complementary receptor domains. In this work, we have directly explored the molecular composition of the minimal functional unit of an agonist ligand and the wild-type G protein-coupled cholecystokinin (CCK) receptor, using photoaffinity labeling with a CCK analogue probe incorporating dual photolabile benzoylphenylalanine (Bpa) residues as sites of covalent attachment. This probe, 125I-D-Tyr-Gly-[(Nle28, 31, Bpa29,33)CCK-26-33], was shown to represent a full agonist and to specifically label the CCK receptor. Like probes incorporating individual photolabile residues in these positions,1,2 the two Bpa residues in the dual photoprobe covalently labeled receptor domains in the amino-terminal tail outside TM1 and in the third extracellular loop outside TM7. Absence of demonstrable receptor dimerization after the establishment of dual sites of covalent attachment supports the presence of these two domains within a single receptor molecule. Demonstration of the covalent adduct of a single probe molecule with the two cyanogen bromide fragments of the CCK receptor representing the expected domains further supports this interpretation. Thus, while domain-swapped dimerization of G protein-coupled receptors may be possible as a mechanism of rescue for nonfunctional molecules, it is not necessary for ligand binding and initiation of signaling at a wild-type receptor in this superfamily. The functional unit for CCK action is normally a ligand-receptor monomer.

Structural differences between HLA-DQ molecules associated with myasthenia gravis characterized by molecular modeling.

Myasthenia gravis (MG) is characterized by muscle weakness due to autoimmunity against the nicotinic acetylcholine receptor (nAChR). MG is associated with polymorphisms in HLA-DQ genes and the aim of the present study was to characterize structural differences in the peptide binding groove of HLA-DQ molecules positively and negatively associated with MG. Three dimensional models of the positively associated DQ2 (DQB1*02) and negatively associated DQ6 (DQB1*0603) molecules were constructed by homology modeling techniques. The differences in peptide binding properties were primarily localized to peptide-anchor pockets P7 and P9, which might be of importance for the binding of disease-associated peptides from the nAChR.

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Molecular modeling of eluted peptides from DQ6 molecules (DQB1*0602 and DQB1*0604) negatively and positively associated with type 1 diabetes.

Insulin-dependent diabetes mellitus (IDDM) is positively associated with HLA-DQ8, DQ2, and DQ6 (B*0604) and negatively with DQ6 (B*0602). The mechanisms by which the DQ molecules control the development of IDDM is not known. DQ6 (B*0602) and DQ6 (B*0604) molecules share the same DQalpha chain but differ in the beta chain by six residues at positions 9, 30, 57, 70, 86, and 87. The aim of the study was to sequence the peptides eluted from both DQ6 molecules and to determine the binding motifs and construct peptides for docking them into the DQ6 peptide binding groove by molecular modeling. EBV transformed B cell line homozygous for DQ6 and hybridoma cell line secreting DQ6 specific antibody were grown in large-scale culture. The DQ6 molecules were precipitated with solid-phase bound antibodies specific for DQ6. The dissociation of peptides from MHC was done with ultrafiltration and separation of peptides by reversed-phase HPLC, using Edman degradation. A special application of Edman degradation is pool sequencing. This approach allowed us to determine common characteristics of all peptides associated with a given MHC molecule. The precipitation of DQ6 molecules and the peptide elution were done successfully. The sequencing of the peptides from DQ6 (B*0602) identified three fractions: (1) IINEPTAAAIAYGLD (Bovine HSP70), (2) IINEPTAAAIAGLDR (Human HSP70), and (3) NPRDAKACVVHGSDLK (Na+/K+ ATPase). Peptide eluted from DQ6 (B*0604) had a sequence ADLFRGTLDPVEK with sequence homology to HSP70 (residues 307-319). We were able to predict the motifs for DQ6 from the ligands eluted. We used molecular modeling as a tool to identify plausible binding motifs for peptides. Our studies show that peptide ADLFRGTLDPVEK and NPRDAKACVVHGSDLK fit well in the respective DQ6 binding grooves. These predicted motifs should then be useful for screening of autoantigens associated with diabetes and identifying the epitopes that are likely to interact with T cells.

Modeling complex molecular interactions involving proteins and DNA.

We have presented a perspective of progress in three areas of simulations of complex molecules: the development of force fields for molecular simulation; the application of computer graphics, molecular mechanics and molecular dynamics in simulations of DNA and DNA-drug complexes and the application of computer graphics, molecular mechanics and quantum mechanics in studies of enzyme substrate interactions. It is our perspective that improvements are being made in force fields, and these will allow a more accurate simulation of structures and energies of complex molecules. In the area of DNA molecular mechanics and dynamics, it is clear that the use of computer graphics model building combined with NMR NOE data is a potentially very powerful tool in accurately determining structures of drug-DNA complexes using molecular mechanics and dynamics. Finally, we are in a position to reasonably simulate structures and (qualitatively) energies for complete reaction pathways of enzymes using a combination of computer graphics, molecular mechanics and quantum mechanics. More accurate energies and pathways are sure to follow, using the combined molecular mechanics/quantum mechanics optimization developed by Singh and the free energy perturbation methods pioneered in Groningen and Houston.

A combined 2D-NMR and molecular dynamics analysis of the structure of the actinomycin D: d(ATGCAT)2 complex.

We present a comparative analysis of an NMR experiment and molecular and harmonic dynamics simulations of an actinomycin D: d(ATGCAT)2 complex. A comparison of NOE measurements and 1/R6 weighted proton-proton distances confirm the general correctness of the Actinomycin D-DNA model proposed by Sobell. There are, however, some substantial differences between the proton-proton distances inferred from the NOE results and the molecular and harmonic dynamics simulations. The remaining discrepancies could either come from contributions of other conformations to the average properties of the complex or from uncertainties in the NMR distance analysis. An analysis of the molecular dynamics helix properties, sugar puckers, hydrogen bonding, rms fluctuations and torsional properties are qualitatively consistent with those from previous simulations, but the presence of an intercalated drug leads to some new structural and dynamical features.

Molecular simulation and drug design.

Computer simulation techniques have become a major tool in the analysis of biomolecular properties and behaviour. These techniques are used extensively in drug and protein design projects, because they can provide information that is complementary to experimental data. Molecular mechanics methods such as energy minimization and molecular dynamics are among the most commonly used simulation techniques for the study of biomolecules. We present here a brief description of some molecular mechanics methods and a few applications examples for biomacromolecules and biomolecular complexes.

Computational biology instruction at the University of Washington Center for Bioengineering.

Computational tools are rapidly becoming an essential component of molecular biology research. However, there is as yet relatively little attention paid to computational biology in the standard curricula of most biology programs. We describe here some of the graduate computational biology courses we have developed in the Center for Bioengineering at the University of Washington, with special emphasis on instructional methods and approaches that appear to work well.

Computer simulation study of the binding of an antiviral agent to a sensitive and a resistant human rhinovirus.

Molecular dynamics simulations have been used to study the free energy of binding of an antiviral agent to the human rhinovirus HRV-14 and to a mutant in which a valine residue in the antiviral binding pocket is replaced by leucine. The simulations predict that the antiviral should bind to the two viruses with similar affinity, in apparent disagreement with experimental results. Possible origins of this discrepancy are outlined. Of particular importance is the apparent need for methods to systematically sample all significant conformations of the leucine side chain.

A molecular mechanical study of complexes formed between 4-nitroquinoline-N-oxide and dinucleoside phosphates.

Molecular mechanical calculations were done on complexes of 4-nitroquinoline-N-oxide (NQO) with various dinucleoside phosphates [(ApT)2, (CpG)2, (GpC)2, and (TpA)2]. Models built using proflavine (uniform C3' endo sugar puckers) and acridine orange (mixed C3' endo (3'-5') C2' endo sugar puckers) dinucleoside phosphate X-ray structures were used in the calculations. Relative binding energies, complex geometries, and various intercalator orientations in the complexes were studied. The results suggest qualitatively different geometries for pyr-(3'-5')-pur and pur-(3'-5')-pyr sequences. Specifically, we find marked distortion in some of the complexes (i.e. there is not a parallel coplanar relationship between the base pairs and intercalator), distortion of the NQO nitro group from planarity in the complexes and mobility of NQO in the intercalation site. We suggest that experimental studies of NQO-dinucleoside phosphate complexes may reveal intercalation complexes which deviate substantially more from a nearly parallel coplanar arrangement of bases and intercalator than has been previously observed.

Use of T cell receptor/HLA-DRB1*04 molecular modeling to predict site-specific interactions for the DR shared epitope associated with rheumatoid arthritis.

OBJECTIVE: To use molecular modeling tools to analyze the potential structural basis for the genetic association of rheumatoid arthritis (RA) with the major histocompatibility complex (MHC) "shared epitope," a set of conserved amino acid residues in the third hypervariable region of the DRbeta chain. METHODS: Homology model building techniques were used to construct molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor (TCR) from T cell clone EM025, which is specific for DR4 molecules containing the shared epitope sequence. Interactive graphics techniques were used to orient the TCR on the DR molecule, guided by surface complementarity analysis. RESULTS: The predicted TCR-MHC-peptide complex involved multiple interactions and specificity for the shared epitope. TCR residues CDR1beta D30, CDR2beta N51, and CDR3beta Q97 were positioned to potentially participate in hydrogen bond interactions with the shared epitope DRbeta residues Q70 and R71. CONCLUSION: These results suggest a structural mechanism in which specific TCR recognition and possibly Vbeta selection are directly influenced by the disease-associated MHC polymorphisms.

Theoretical calculation of relative binding affinity in host-guest systems.

The relative free energy of binding the anions Cl- and Br- to the macrotricyclic receptor SC24 in water has been computed by a computer simulation technique. This result and an incidental result for the relative free energy of hydration of the anions are in excellent agreement with experimental data. The simulation approach to ligand-receptor interactions that is described here has significant potential as a predictive tool in chemistry, biochemistry, and pharmacology.

MD Display: an interactive graphics program for visualization of molecular dynamics trajectories.

MD Display was developed as a means of visualizing molecular dynamic trajectories generated by Amber. 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.

A structural model for TCR recognition of the HLA class II shared epitope sequence implicated in susceptibility to rheumatoid arthritis.

HLA molecules associated with rheumatoid arthritis (RA) contain a discrete structural element known as the shared epitope, a set of conserved amino acid residues located on the alpha helical portion of the class II beta chain. Each of the different HLA molecules associated with RA contain the same shared epitope sequence, although they may vary markedly in other regions of the class II structure, which also determine peptide-class II interactions. Previous mutagenesis studies and structural modelling indicate that key polymorphic amino acid side chains within the shared epitope sequence are in locations likely to contact the T cell receptor (TCR) during the trimolecular activation reaction between the HLA-peptide complex and TCR. We have evaluated the potential structural basis for such shared epitope recognition by analysing detailed molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor from T cell clone EM025, specific for HLA-DR4 molecules which carry the shared epitope. A likely orientation for the trimolecular complex was deduced in which the EM025 alpha chain interacts with the DR alpha chain and the EM025 beta chain interacts with the DR beta chain; residues Q70 and R71 within the DR beta chain shared epitope region are positioned for hydrogen bond interactions directly with Q97 of the TCR beta CDR3 region, D30 of the TCR beta CDR1 region, and possibly N51 of the TCR beta CDR2 region, indicating a degree of specific selection and interaction which encompasses multiple TCR contacts. These findings suggest a structural basis for the genetic associations with the HLA shared epitope and the potential contribution of this region to oligoclonal T cell selection and expansion in RA.