Stanley M. Swanson

Determination of the structure of an endoglucanase from Aspergillus niger and its mode of inhibition by palladium chloride.

The fungus Aspergillus niger is a main source of industrial cellulase. beta-1,4-Endoglucanase is the major component of cellulase from A. niger. In spite of widespread applications, little is known about the structure of this enzyme. Here, the structure of beta-1,4-endoglucanase from A. niger (EglA) was determined at 2.1 A resolution. Although there is a low sequence identity between EglA and CelB2, another member of family 12, the three-dimensional structures of their core regions are quite similar. The structural differences are mostly found in the loop regions, where CelB2 has an extra beta-sheet (beta-sheet C) at the non-reducing end of the binding cleft of the native enzyme. Incubation of EglA with PdCl(2) irreversibly inhibits the EglA activity. Structural studies of the enzyme-palladium complex show that three Pd(2+) ions bind to each EglA molecule. One of the Pd(2+) ions forms a coordinate covalent bond with Met118 S(delta) and the nucleophilic Glu116 O(epsilon1) at the active site of the enzyme. The other two Pd(2+) ions bind on the surface of the protein. Binding of Pd(2+) ions to EglA does not change the general conformation of the backbone of the protein significantly. Based on this structural study, one can conclude that the palladium ion directly binds to and blocks the active site of EglA and thus inactivates the enzyme.

ANALYSIS OF THE STRUCTURE OF HIV-1 PROTEASE COMPLEXED WITH A HEXAPEPTIDE INHIBITOR. PART II: MOLECULAR DYNAMIC STUDIES OF THE ACTIVE SITE REGION

Six models of the catalytic site of HIV‐1 protease complexed with a reduced peptide inhibitor, MVT‐101, were investigated. These studies focused on the details of protonation of the active site, its total net charge and hydrogen bonding pattern, which was consistent with both the observed coplanar configuration of the acidic groups of the catalytic aspartates (Asp‐25 and Asp‐125) and the observed binding mode of the inhibitor. Molecular dynamic simulations using AMBER 4.0 indicated that the active site should be neutral. The planarity of the aspartate dyad may be due to the formation of two hydrogen bonds: one between the inner Oδ1oxygen atoms of the two catalytic aspartates and another between the Oδ2atom of Asp‐125 and the nitrogen atom of the reduced peptide bond of the bound inhibitor. This would require two additional protonations, either of both aspartates, or of one Asp and the amido nitrogen atom of Nle‐204. Our results favor the Asp‐inhibitor protonation but the other one is not excluded. Implications of these findings for the mechanism of enzymatic catalysis are discussed. Dynamic properties of the hydrogen bond network in the active site and an analysis of the interaction energy between the inhibitor and the protease are presented.

Mapping the atomic environment of functional groups: turning 3D scatter plots into pseudo-density contours

Abstract A 3D map of the atomic environment about a pharmacophore, or any other functional group, can be constructed by combining data from crystal structures containing that group. 3D scatter plots show the positions where nonbonded atoms interact with the group. By placing a ‘smearing’ function at each of these points, we convert the scatter plot into a density map. Contours at various densities reveal non-uniformities in the distribution, which may indicate preferred directions of chemical interaction. Quantitative comparisons between density maps are easier to make than between scatter plots. We illustrate the method by examining the distribution of anions about the trimethylammonium group, a substructure of the neurotransmitter acetylcholine.

Core Tracing: Depicting Connections Between Features in Electron Density

Core tracing is a threshold-independent method of determining connectivity (long chains of high-density values) in electron-density maps. It gives visually sparse pictures of large volumes which are useful for initial fitting and for molecular-boundary determination. New methods for visual presentation of the traces are suggested by the way that the connectivity is parameterized in terms of local connections between maxima and the saddle (lowest) points along the connecting paths. The algorithm also partitions the density into small compact volumes containing the maxima. These volumes are useful for localization and statistical analysis.

Intermolecular enzyme-ligand animation in the active site of porcine pancreatic elastase with acetyl-alanine-proline-alanine by means of molecular dynamics calc

Abstract An interactive molecular display system has been developed to enable the visualization of the 540 conformations derived from a molecular dynamics calculation of the fluctuations of the enzyme-ligand complex formed between porcine pancreatic elastase (PPE) and acetyl-alanine-proline-alanine (APA). Dynamical interactions between the receptor and the inhibitor are observed at the active site, e.g. the pyrrolidone ring of the ligand 2-proline residue is observed to flex via restrained dihedral angle rotations; the terminal acetate moiety is seen to move between two adjacent binding loci. An animated molecular graphics display, linked to a molecular or stochastic dynamics method, is an instructive and predictive tool for investigating dynamical interactions of enzyme-ligand binding.

Simulations of dynamical properties of a Michaelis complex: porcine pancreatic elastase and the hexapeptide, Thr-Pro-n Val-Leu-Tyr-Thr.

Molecular dynamic simulations (30ps) of the Michaelis complex of hexapeptide (Thr-Pro-nVal-Leu-Tyr-Thr) bound to porcine pancreatic elastase (PPE) hydrated by about 2000 water molecules have been performed using the AMBER 3.0 program package. Dynamical properties of the conformation of the active site have been examined. A comparison with previously reported simulations of native PPE shows that after the substrate is bound, the catalytically crucial H-bond between O gamma-H group of (Ser 195) and nitrogen N epsilon (His 57) is more readily formed. These results show, however, that the H-bond does not adopt the most favorable conformation. The O gamma-H group of Ser 195 has a statistical preference for an attractive interaction with the O = C carbonyl (Ser 214) rather than the nitrogen N epsilon (His 57).

Dynamical analysis of the conformation of the active site of porcine pancreatic elastase in native and Michaelis complex states. Molecular dynamics simulations

Abstract Mechanistic details of events preceeding and occurring during enzymatic catalysis are extremely difficult to obtain experimentally. While molecular dynamics methods permit the simulation of discrete steps along the catalytic pathway, they also can overwhelm the investigator with a mountain of structural details, necessitating the development of specific analytic tools. Based on recent crystallographic data, several molecular dynamics simulations have been performed using the AMBER 3.0 program package. Two different simulations for the hydrated native enzyme (100 and 50 ps) and two simulations (30 and 70 ps) of the Michaelis complex of this enzyme with the substrate (Thr-Pro-nVal-Leu-Tyr-Thr) have been performed. Dynamical properties of the active site (especially the catalytic tetrad: Ser-195, His-57, Asp-102 and Ser-214; chymotrypsinogen numbering system) have been examined using the program MD_ANALYSIS_1. It, together with the program MDKINO, facilitates analysis of dynamical changes of conformation (especially the hydrogen bond network) of the active site. Hydrogen bonding among Asp-102, Ser-214 and His-57 was quite stable, but the catalytic Ser-195 sidechain was flexible. Therefore the catalytically crucial H-bond between HO γ (Ser-195) and N ϵ (His-57) is relatively labile. In the native case (i.e. without substrate) this H-bond was never formed due to competition for the acceptor atom (N ϵ ) between water molecules and the HO γ group. In the Michaelis complex the H-bond is more readily formed, although the sidechain (Ser-195) may sometimes change its conformation do to the influence of the carbonyl group of Ser-214. Due to the dynamical motion of the enzyme there were six different short periods in which the distance between both heavy atoms in this crucial H-bond was less than 2.6 A, which may facilitate proton transfer from Ser-195 to His-57 (the first step during the proper catalysis). These results suggest mechanistic details about the precise clockwork of a functioning enzyme.

Alternative electron density models for structural biochemistry

Abstract Computer graphics can depict intrinsically three-dimensional structures. Restriction to plane sections of an electron density, or even to the widely used cagecontouring technique is not necessary. By detecting two-dimensional and three-dimensional density maxima, the algorithm discussed here offers new possibilities for describing the trace of linked density maxima. The core-tracing technique is described and illustrated by means of stereo views.

The Effect of Noise on Entropy

A complementary relationship between the entropy (S) and the variance (sigma(2)) of an electron-density map is derived by approximating the logarithmic term in the entropy expression by a series expansion around the average map density. The resulting expression is S approximately ln N - 1/2sigma(2), where N is the number of grid points and sigma is the r.m.s. deviation from the mean in a map normalized to unit mean. The algebraic expression is of interest because it is consistent with and allows numerical evaluation of the surprising argument that noise decreases the entropy of a map. The argument is that a noise contribution by itself generates a certain variance that is independent of the atomic structure and that adds to the variance due to the structure. Increased variance corresponds to decreased entropy. This property of noise provides an intuitively reasonable justification for maximizing the entropy of an electron-density map in the quest for more readily interpretable maps of macromolecules. The entropy-variance relationship also extends the range of applicability of the entropy concept to maps with a limited amount of negative density. The approximation which leads to the entropy-variance relationship is most applicable where it is most likely to be useful - in experimental maps of relatively low structure definition.