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The binding of NAD to liver alcohol dehydrogenase has been studied in four different ternary complexes by using crystallographic methods. These complexes crystallize isomorphously in a triclinic crystal form which contains the whole dimer of the enzyme in the asymmetric unit. This form of the enzyme has been refined at 2.9-A resolution to a crystallographic R factor of 0.22. NAD binds in essentially the same way in these complexes. The binding site is located at the central part of the coenzyme binding domain. The adenine ring binds with hydrophobic interactions between two isoleucine side chains. Both ribose rings have 2E(C2'-endo) puckering, and each ribose makes three hydrogen bonds to the enzyme. The pyrophosphate bridge has hydrogen bonds to the side chains of arginine-47 and -369 and to main chain nitrogen atoms from the amino ends of two alpha-helices. The nicotinamide ring is in van der Waals contact with the active-site zinc atom and with the sulfur atoms of its cysteine ligands. The carboxamide group is about 30 degrees out of the plane of the nicotinamide ring and hydrogen bonds to main chain atoms of residues 292,317, and 319. The overall conformation of the NAD molecule is similar to that observed for other dehydrogenases, but differs in details. In the presence of the coenzyme, the enzyme undergoes a large conformational change from an open to a closed form. This conformational change has three major effects: to create favorable binding interactions with groups of the enzyme, to enclose the coenzyme and gain binding energy for the coenzyme by reducing the accessible surface area, and to close off one entrance to the active site. As a comparison, ADP-ribose binding has been studied in the open form of the enzyme. The adenosine moiety binds in a similar way as NAD, while the rest of the molecule has different interactions.

Human serum retinol binding protein (RBP) in complex with retinol has been crystallographically refined to an R-factor of 18.1% with 2A resolution data. The protein topology results in an anti-parallel beta-barrel that encapsulates the retinol ligand. A detailed description of the protein and the binding site is provided. Our structural work has helped to define a family of proteins, many of which are carrier proteins for smaller ligand molecules. We describe the structural basis for the conservation of sequence within the family.

The solution conformation of the antibacterial polypeptide cecropin A from the Cecropia moth is investigated by nuclear magnetic resonance (NMR) spectroscopy under conditions where it adopts a fully ordered structure, as judged by previous circular dichroism studies [Steiner, H. (1982) FEBS Lett. 137, 283-287], namely, 15% (v/v) hexafluoroisopropyl alcohol. By use of a combination of two-dimensional NMR techniques the 1H NMR spectrum of cecropin A is completely assigned. A set of 243 approximate interproton distance restraints is derived from nuclear Overhauser enhancement (NOE) measurements. These, together with 32 distance restraints for the 16 intrahelical hydrogen bonds identified on the basis of the pattern of short-range NOEs, form the basis of a three-dimensional structure determination by dynamical simulated annealing [Nilges, M., Clore, G.M., & Gronenborn, A.M. (1988) FEBS Lett. 229, 317-324]. The calculations are carried out starting from three initial structures, an alpha-helix, an extended beta-strand, and a mixed alpha/beta structure. Seven independent structures are computed from each starting structure by using different random number seeds for the assignments of the initial velocities. All 21 calculated structures satisfy the experimental restraints, display very small deviations from idealized covalent geometry, and possess good nonbonded contacts. Analysis of the 21 converged structure indicates that there are two helical regions extending from residues 5 to 21 and from residues 24 to 37 which are very well defined in terms of both atomic root mean square differences and backbone torsion angles. For the two helical regions individually the average backbone rms difference between all pairs of structures is approximately 1 A. The long axes of the two helices lie in two planes, which are at an angle of 70-100 degrees to each other. The orientation of the helices within these planes, however, cannot be determined due to the paucity of NOEs between the two helices.

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