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Realistic representation of protein flexibility in biomolecular simulations remains an unsolved fundamental problem and is an active area of research. The high flexibility of the cytochrome P450 2D6 (CYP2D6) active site represents a challenge for accurate prediction of the preferred binding mode and site of metabolism (SOM) for compounds metabolized by this important enzyme. To account for this flexibility, we generated a large ensemble of unbiased CYP2D6 conformations, to which small molecule substrates were docked to predict their experimentally observed SOM. SOM predictivity was investigated as a function of the number of protein structures, the scoring function, the SOM-heme cutoff distance used to distinguish metabolic sites, and intrinsic reactivity. Good SOM predictions for CYP2D6 require information from the protein. A critical parameter is the distance between the heme iron and the candidate site of metabolism. The best predictions were achieved with cutoff distances consistent with the chemistry relevant to CYP2D6 metabolism. Combination of the new ensemble-based docking method with estimated intrinsic reactivities of substrate sites considerably improved the predictivity of the model. Testing on an independent set of substrates yielded area under curve values as high as 0.93, validating our new approach.

Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 A resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulfur atoms of IGF-1. The structure reveals a well-defined conformation for much of the C-region, which extends away from the core of IGF-1 and has residues known to be involved in receptor binding prominently displayed in a type II beta-turn. In the crystal, these residues form a dimer interface, but analytical ultracentrifugation experiments demonstrate that at physiological concentrations IGF-1 is monomeric. A single detergent molecule contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions. Biophysical and biochemical data show that the detergent binds to IGF-1 specifically and blocks binding of IGFBP-1 and IGFBP-3.

A theoretical study of the alkylation reaction of toluene with methanol catalyzed by the acidic Mordenite (Si/Al = 23) is reported. Cluster DFT as well as periodical structure DFT calculations have been performed. Full reaction energy diagrams of the elementary reaction steps that lead to the formation of the three xylene isomers are given. The use of periodical structure calculations allows one to account for zeolite framework electrostatic contributions and steric constraints that take place in zeolitic catalysts. Especially the steric constraint energy contribution has a significant effect on the energies and bond formation paths. The activation energy barrier of p-xylene formation is found to be approximately 20 kJ/mol lower than the corresponding values for the formation of its isomers. Computed host-guest binding energies according to the DFT method need a correction due to the absence of the dispersive interaction with the zeolite wall. Apparent activation energies obtained with this correction are in good agreement with experimental data.

Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells [Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690]. Here we describe the structure of Apo2L at 1.3 A resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.

Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and, therefore, a prime therapeutic target for the development of antagonists for the treatment of cancer. As a first step toward this goal, phage display was used to generate peptides that bind to the receptor-binding domain (residues 8-109) of VEGF and compete with receptor [Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y. K., Wells, J. A., and Cunningham, B. C. (1999) Biochemistry 38, 17754-17764]. The crystal structure of VEGF in complex with one of these peptides was solved and refined to a resolution of 1.9 A. The 20-mer peptide is unstructured in solution and adopts a largely extended conformation when bound to VEGF. Residues 3-8 form a beta-strand which pairs with strand beta6 of VEGF via six hydrogen bonds. The C-terminal four residues of the peptide point away from the growth factor, consistent with NMR data indicating that these residues are flexible in the complex in solution. In contrast, shortening the N-terminus of the peptide leads to decreased binding affinities. Truncation studies show that the peptide can be reduced to 14 residues with only moderate effect on binding affinity. However, because of the extended conformation and the scarcity of specific side-chain interactions with VEGF, the peptide is not a promising lead for small-molecule development. The interface between the peptide and VEGF contains a subset of the residues recognized by a neutralizing Fab fragment and overlaps partially with the binding site for the Flt-1 receptor. The location of the peptide-binding site and the hydrophilic character of the interactions with VEGF resemble more the binding mode of the Fab fragment than that of the receptor.

Mutational studies have shown that over two-thirds of the contact side chains at the human growth hormone (hGH)-receptor interface have little or no impact on binding affinity when converted to alanine [Cunningham, B. C., & Wells, J. A. (1993) J. Mol. Biol. 234, 554-563; Clackson, T., & Wells, J. A. (1995) Science 267. 383-386]. Herein, three of the most buried, yet functionally inert, residues on hGH (F25, Y42, and Q46) have been simultaneously mutated to alanine. Binding kinetics of the triple-alanine mutant shows that neither association nor dissociation rates are significantly affected and only slight, local disorder is seen in the crystal structure. However, large and compensating changes were observed in the enthalpy and entropy of binding as determined by isothermal titration calorimetry. The triple-alanine mutant bound with a more favorable enthalpy (delta H = -12.2 +/- 0.7 kcal/mol) and corresponding less favorable entropy [delta S = -2.3 +/- 2.4 cal/(mol.K)] compared to the wild-type interaction [delta H = -9.4 +/- 0.3 kcal/mol; delta S = 7.7 +/- 1.2 cal/(mol.K)]. Dissection of the triple-alanine mutant into the single F25A and double Y42A/Q46A mutant showed that the more favorable enthalpy was derived from the removal of the F25 side chain on helix-1 of the hormone. The delta Cp values for both the triple-alanine mutant [-927 +/- 10 cal/(mol.K)] and the individual mutants were significantly more negative than the delta Cp for the wild-type interaction [-767 +/- 34 cal/(mol.K)]. Such negative delta Cp values are consistent with the proposal that the hydrophobic effect is the primary contributor to the free energy of binding at this protein-protein interface. These results show that multiple-alanine mutations at contact residues may not affect binding kinetics, affinity, or global structure; however, they can produce local structural changes and can cause large compensating effects on the heat and entropy of binding. These studies emphasize that one cannot infer binding free energy from the existence of contacts alone and further support the notion that only a small set of contacts are crucial for the human growth hormone-receptor interaction.

Tissue factor, the obligate cofactor for coagulation factor VII, plays an essential role in hemostasis by initiating the extrinsic pathway of blood coagulation upon vascular damage, making it a promising target for new anticoagulant therapies in the treatment of thrombosis and sepsis. The three-dimensional structure of the extracellular domain of tissue factor, determined by X-ray crystallography at a resolution of 2.4 A, consists of two domains of approximately equal size, with a topology characteristic of fibronectin type III modules. Comparison of tissue factor with the extracellular domain of the growth hormone receptor, which belongs to the same receptor superfamily, shows that the relative orientation between these domains as well as the domain-domain interface is very different. These differences have dramatic consequences for the residues in tissue factor that are homologous to the binding determinants of the growth hormone receptor. Alanine-scanning mutagenesis has identified tissue factor residues important for factor VIIa binding. The structure shows that the binding site is located in the domain-domain interface region but on the opposite side of the molecule compared to the growth hormone receptor, with the binding determinants residing on beta-strands rather than on loops.

The structure of Ca-prothrombin fragment 1 (residues 1-156 prothrombin) has been solved and refined at 2.2-A resolution by X-ray crystallographic methods. The first two-thirds of the Gla domain (residues 1-48) and two carbohydrate chains (approximately 5 kDa) are disordered in crystals of apo-fragment 1. When crystals are grown in the presence of Ca2+ ions, the Gla domain exhibits a well-defined structure binding seven Ca2+ ions, but the carbohydrate is still disordered. Even so, the crystallographic R factor reduced to 0.171. The folding of the Gla domain is dominated by 9-10 turns of three different alpha-helices. These turns produce two internal carboxylate surfaces composed of Gla side chains. A polymeric array of five Ca2+ ions separated by about 4.0 A intercalates between the carboxylate surfaces. The coordination of the Ca2+ ions with Gla carboxylate oxygen atoms and water molecules leads to distorted polyhedral arrangements with mu-oxo bridges in a highly complex array that most likely orchestrates the folding of the domain. The overall mode of interaction of the Ca2+ ions is new and different from any Ca2+ ion-protein interactions heretofore observed or described. The fluorescence quenching event observed upon Ca2+ ion binding is due to a disulfide-pi-electron interaction that causes a 100 degrees reorientation of Trp42 of the Gla domain. The Ca2+ ion interaction also affords the N-terminus protection from acetylation because the latter is buried in the folded structure and makes hydrogen-bonding salt bridges with Gla17, Gla21, and Gla27. The Gla domain and its trailing disulfide unit associate intimately and together give rise to a domain-like structure. Electrostatic potential calculations indicate that the Gla domain is very electronegative. Since most of the carboxylate oxygen atoms of Gla residues are involved in Ca2+ ion binding, leaving only a few for bridging Ca2+ ion-phospholipid interactions, the role of bridging Ca2+ ions might be generally unspecific, with Ca2+ ions simply intervening between the negative Gla domain and negative head groups of the membrane surface. The folding of the kringle structure in apo- and Ca-fragment 1 is essentially the same. However, the Ser36-Ala47 helix of the Gla domain pivots around Cys48, shifting by approximately 30 degrees, and the helix encroaches on the kringle producing some concomitant changes. These might be related to the protection of carbohydrate carrying Asn101 from acetylation in the Ca-fragment 1 structure.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.