X-ray studies in molecular interactions involving amino acids

Abstract

Non-covalent interactions play a crucial role in the structure, assembly, and function of proteins. A program of systematic X-ray studies on complexes involving amino acids and short peptides, among themselves as well as with other biomolecules, is being carried out in this laboratory in an attempt to elucidate, at atomic resolution, the possible geometrical features of such interactions. The author's contribution to this ongoing program forms a major part of the thesis. A brief survey of the earlier work on complexes carried out in this laboratory is therefore presented in the introductory chapter. Also surveyed is the work done elsewhere on complexes between amino acids and their derivatives on the one hand, and nucleic acid constituents on the other.

The rate-limiting, and perhaps the most difficult, step in the X-ray studies on crystalline complexes is the preparation of suitable single crystals. Chapter 2 describes the crystallization experiments-successful as well as unsuccessful-carried out by the author. Crystals of the following complexes suitable for X-ray studies could be grown:

(a) L-arginine L-aspartate (b) L-ornithine L-aspartate (c) N-acetyl glycyl L-lysine methyl ester acetate (d) L-lysine D-pantothenate

The space group and the unit cell dimensions of the crystals were determined from oscillation and Weissenberg photographs.

The X-ray crystal structure analysis of L-arginine L-aspartate is reported in Chapter 3. The structure (space group, P2 , Z = 2) was solved by direct methods and refined to a final R value of 0.044 for 1226 independent counter-measured reflections. The conformation of the arginine molecule is different from those previously observed, whereas the conformation of the aspartate ion is similar to that found in L-aspartic acid, DL-aspartic acid, and L-lysine L-aspartate. The unlike molecules aggregate into separate alternating layers, and the -amino and the -carboxylate groups in the arginine layer are periodically brought into close proximity in a “head-to-tail” arrangement. There exists a specific ion-pair interaction involving electrostatic attraction and two nearly parallel N-H···O hydrogen bonds between the guanidyl group and the -carboxylate group of the aspartate ion.

Chapter 4 describes a systematic study of specific interactions involving the guanidyl group observed in crystal structures. The guanidyl group, which forms part of the arginine side chain, is capable of taking part in four different types of specific interactions with carboxylate, phosphate, and other groups. Two of these specific interactions involve two nearly parallel N-H···O hydrogen bonds, while each of the remaining two involves converging hydrogen bonds with a common acceptor. A very high frequency of occurrence of these specific interactions, especially those involving two nearly parallel hydrogen bonds, is observed in crystal structures containing arginine or monoalkyl guanidines, indicating the intrinsic propensity of the guanidyl group to take part in such interactions.

Chapter 5 deals with the structure of L-ornithine L-aspartate hemihydrate. The structure (space group, C2°, Z = 4) solved by direct methods was refined to an R value of 0.041 for 1270 observed reflections. The conformation of the two amino acid molecules in the structure is somewhat different from those observed in other crystal structures containing them. The crystal structure is stabilized by ionic interactions accompanied by hydrogen bonds. The unlike molecules aggregate into separate two-fold helices; each helix of one type is surrounded by, and is in hydrogen-bonded contact with, four helices of the other type. The arrangement of molecules in the structure is such that it can be described as consisting of alternating hydrophilic and hydrophobic regions. The hydrophilic regions contain hydrogen-bonded loops, each made up of two amino groups and two carboxylate groups. The structure also provides the first example of a head-to-tail sequence involving two types of amino acids.

The crystal structure of the acetate of the end-protected dipeptide N-acetyl glycyl L-lysine methyl ester is reported in Chapter 6. The structure (space group, P2 2 2 ; Z = 4) was solved again using direct methods and refined to R = 0.079 for 993 observed reflections. The fully extended lysine side chain in the molecule is staggered between the main chain amino and carbonyl groups. The dipeptide molecules in the crystal structure are arranged in two-fold helices centered on 2 screw axes. These helices are interconnected through interactions involving the acetate and the side chain amino groups. Each acetate group bridges two adjacent side chain amino groups.

Chapter 7 reports the crystal structure of L-lysine D-pantothenate. The structure (space group, F2 , Z = 2), the solution of which turned out to be less than straightforward, was refined to an R value of 0.053 for 1868 reflections. The zwitterionic positively charged lysine molecules in the structure assume the sterically most favorable conformation with an all-trans side chain trans to the -carboxylate. The pantothenate anion has a somewhat folded conformation stabilized by an intramolecular bifurcated hydrogen bond. The unlike molecules aggregate into separate alternating layers. The molecules in the lysine layer form a head-to-tail sequence parallel to the a-axis. Among the interactions which hold the adjacent layers together, those involving the side chain amino group of lysine and the carboxylate group in the pantothenate anion are of particular interest.

The conformational analysis of D-pantothenic acid using classical semiempirical methods is described in the final chapter. The pantothenic acid molecule can exist in the neutral form (I) or in the ionized form (II) with a deprotonated negatively charged carboxyl group. The neutral molecule as well as the anion is highly flexible and has an ensemble of several allowed conformations rather than one or two unique conformations. The possibility of intramolecular hydrogen bonding in different allowed conformations has also been explored. A bifurcated hydrogen bond involving a carboxyl (or carboxylate) atom and a hydroxyl oxygen atom, as acceptors, and the amide nitrogen atom as the donor occurs frequently in both I and II. Among the two crystal structures containing pantothenic acid reported so far, the conformation of the molecule in L-lysine D-pantothenate lies in the allowed region and is stabilized by bifurcated intramolecular hydrogen bonds, whereas that in the CaBr salt falls in a disallowed region, presumably due to the requirements of metal coordination.

The author has been deeply involved in the ongoing efforts in this laboratory in the area of macromolecular crystallography. His contribution in this area, which consisted primarily of the crystallization and the preliminary X-ray studies of two forms of the anti-T lectin from peanut (Arachis hypogaea), is described in an appendix. The form which crystallizes at neutral pH is orthorhombic with a tetrameric molecule in the asymmetric unit. The other form, which crystallizes at pH 4.6, is monoclinic, again with four subunits in the asymmetric unit. Two of the subunits in this form, however, appear to be related to the other two by an approximate non-crystallographic twofold axis perpendicular to the monoclinic unique axis.