Marjorie M. Harding

Geometry of metal–ligand interactions in proteins

The geometry of metal-ligand interactions in proteins is examined and compared with information for small-molecule complexes from the Cambridge Structural Database (CSD). The paper deals with the metals Ca, Mg, Mn, Fe, Cu, Zn and with metal-donor atom distances, coordination numbers and extent of distortion from ideal geometry (octahedral, tetrahedral etc.). It assesses the agreement between geometry found in all metalloprotein structures in the Protein Data Bank (PDB) determined at resolution < or = 1.6 A with that predicted from the CSD for ligands which are analogues of amino-acid side chains in proteins [Harding (1999), Acta Cryst. D55, 1432-1443; Harding (2000), Acta Cryst. D56, 857-867]. The agreement is reasonably good for these structures but poorer for many determined at lower resolution (examined to 2.8 A resolution). For metal-donor distances, the predictions from the CSD, with minor adjustments, provide good targets either for validation or for restraints in refinement of structures where only poorer resolution data is available. These target distances are tabulated and the use of restraints is recommended. Validation of angles or the use in refinement of restraints on angles at the metal atom is more difficult because of the inherent flexibility of these angles. A much simplified set of parameters for angle restraints with quite large standard deviations is provided. (Despite the flexibility of the angles, acceptable and preferred coordination numbers and shapes are well established and a summary table is provided.) An unusual and perhaps biochemically important feature of Zn coordination with carboxylate seen in the CSD examples is also clearly present in metalloprotein structures. With metals like Ca, carboxylate coordination is monodentate or bidentate (two M-O bonds of nearly equal length). In Zn carboxylates a continuous range between monodentate and bidentate coordination is found, with one Zn-O bond of normal length and another of any length between this and a van der Waals contact.

X-ray diffraction studies of polysaccharide sulphates: Double helix models for κ- and ι-carrageenans

Abstract Two polysaccharides have been studied which approximate in structure to alternating copolymers, (−A−B) n , in which B is a residue of β- d -galactose-4sulphate and A is a residue of 3,6-anhydro-α- d -galactose (κ-carrageenan) or its 2-sulphate (ι-carrageenan). The glycoside linkages are Al→3B and B1→4A. A small proportion of A residues also occur as α- d -galactose-6-sulphate and 2,6-disulphate in the natural polymers but were removed by fractionation or conversion to the anhydride with alkaline borohydride. X-ray diffraction photographs of oriented fibres of salts with various monovalent cations then showed fibre axis repeat distances of 24.6 A (κ-carrageenan) and 13.0 A (ι-carrageenan). Very similar models can be proposed for both polysaccharides, based on striking relationships between features in the various diffraction photographs, mathematical derivation of all models with appropriate dimensions and symmetry, and calculation of cylindrically averaged Fourier transforms. They are double helices with three disaccharide residues in a complete turn of each single chain, in 24.6 A (κ-carrageenan) or 26.0 A (ι-carrageenan). In ι-carrageenan the second chain is displaced exactly half a pitch from the first.

Metal–ligand geometry relevant to proteins and in proteins: sodium and potassium

In previous papers [Harding (2001), Acta Cryst. D57, 401–411, and references therein] the geometry of metal–ligand interactions was examined for six metals (Ca, Mg, Mn, Fe, Cu, Zn) using the Protein Data Bank and compared with information from accurately determined structures of relevant small-molecule crystals in the Cambridge Structural Database. Here, the environments of Na+ and K+ ions found in protein crystal structures are examined in an equivalent way. Target M+⋯O distances are proposed and the agreement with observed distances is summarized. The commonest interactions are with water molecules and the next commonest with main-chain carbonyl O atoms.

MESPEUS: a database of the geometry of metal sites in proteins

A database with details of the geometry of metal sites in proteins has been set up. The data are derived from metalloprotein structures that are in the Protein Data Bank [PDB; Berman, Henrick, Nakamura & Markley (2006). Nucleic Acids Res. 35, D301–D303] and have been determined at 2.5 A resolution or better. The database contains all contacts within the crystal asymmetric unit considered to be chemical bonds to any of the metals Na, Mg, K, Ca, Mn, Fe, Co, Ni, Cu or Zn. The stored information includes PDB code, crystal data, resolution of structure determination, refinement program and R factor, protein class (from PDB header), contact distances, atom names of metal and interacting atoms as they appear in the PDB, site occupancies, B values, coordination numbers, information on coordination shapes, and metal–metal distances. This may be accessed by SQL queries, or by a user-friendly web interface which searches for contacts between specified types of atoms [for example Ca and carboxylate O of aspartate, Co and imidazole Nδ of histidine] or which delivers details of all the metal sites in a specified protein. The web interface allows graphical display of the metal site, on its own or within the whole protein molecule, and may be accessed at http://eduliss.bch.ed.ac.uk/MESPEUS/. Some applications are briefly described, including a study of the characteristics of Mg sites that bind adenosine triphosphate, the derivation of an average Mg—Ophosphate distance and some problems that arise when average bond distances with high precision are required.

Metals in protein structures: a review of their principal features

Metals are present in more than one-third of all proteins as they occur in nature, and are usually important in biological function or maintenance of the structure. Some are present as cations, directly associated with amino acid functional groups of the protein, others within small molecule cofactors associated with the protein. For the 10 metals commonly found as cations, Na, Mg, K, Ca, Mn, Fe, Co, Ni, Cu and Zn, a survey is given of occurrence, relative frequencies of both metal and donor atom or group type, and geometry of coordination. The survey is based on crystal structure information deposited in the Protein Data Bank (PDB) [Berman, H.; Henrick, K.; Nakamura, H.; Markley, J.L. The Worldwide Protein Data Bank (wwPDB): Ensuring a Single, Uniform Archive of PDB Data. Nucleic Acids Res. 2007, 35, D301–D303]. The precision and reliability of this information is assessed in detail. Illustrative examples are given for each metal, including, usually, details of the structure of the metal site in relation to the whole protein and to its function; there are comparisons between metals and descriptions of features such as binding to carboxylate and multiple metal sites close to each other. Metals found within cofactors which associate with the protein, most notably Mo, are included within these examples. Also included briefly are the prediction of metal sites in proteins resulting from genomic synthesis, information which can be derived from methods other than X-ray diffraction of crystals, and metal–protein systems which do not occur naturally.

The crystal structure of insulin: II. An investigation of rhombohedral zinc insulin crystals and a report of other crystalline forms†

X-Ray diffraction photographs have been taken of zinc-free cubic insulin and of monoclinic zinc insulin crystals, prepared from buffers containing phenol. The wet cubic crystals are very small rhombic dodecahedra, a = 76 A, ρ = 1·09 approx. and space group, I23 or I213; there is one insulin monomer (mol. wt about 6000) in the asymmetric unit. Wet monoclinic crystals have a = 62·3, b = 61·8, c = 47·8 A, β = 110·7°, ρ = 1·19 g cm−3 and air-dried crystals have a = 61·4, b = 53·4, c = 43·8 A, β = 134·5°, ρ = 1·29 g cm−3; the space group is P21 and there are six insulin monomers in the asymmetric unit. A more detailed investigation of two rhombohedral forms with space group R3, has been carried out. Wet 2 Zn insulin crystals, prepared from pig insulin in citrate buffers, have a = 82·5 and c = 34·0 A for the hexagonal unit cell, and ρ = 1·24 g cm−3, implying two insulin monomers per asymmetric unit or six monomers and two zinc atoms per rhombohedral cell. This 2 Zn insulin is essentially the same as rhombohedral zinc insulin formerly prepared from cattle insulin in phosphate buffers and studied by X-ray methods. 4 Zn insulin was prepared from citrate buffers similar except for the addition of sodium chloride in place of acetone. Wet crystals have a = 80·7, c = 37·6 A, ρ = 1·245 g cm−3, and contain six insulin monomers and four zinc atoms per rhombohedral cell. Three-dimensional X-ray intensity data have been collected and Patterson distributions calculated. These show that the molecular arrangement in 2 Zn insulin and 4 Zn insulin is very similar; the difference is of the same order of magnitude as that between wet and air-dried crystals.

LSCALE – the new normalization, scaling and absorption correction program in the Daresbury Laue software suite

A new program in the Daresbury Laue software suite has been developed for the scaling and normalization of Laue intensity data, to yield fully corrected structure amplitudes. Previously available routines have been improved, and additional options for refinement, control and statistical diagnostic output provided. A new feature, namely a wavelength- and position-dependent absorption correction that models a two-dimensional surface derived from the Laue data alone, is discussed in detail; it is tested on simulated and real data, and the improvement in data quality is demonstrated. The wavelength normalization function is now able, when sufficiently redundant experimental data are available, to model fine details such as the features arising from the modification of the incident intensity spectrum by a platinum mirror in the beamline optics. A full data set for tetragonal lysozyme is processed with the new program, and extensive statistical output is given.

The crystal structure of insulin: III. Evidence for a 2-fold axis in rhombohedral zinc insulin

The existence of non-space-group symmetry elements in rhombohedral 2 Zn insulin and 4 Zn insulin crystals has been investigated. A rotation function shows the existence of a 2-fold axis perpendicular to the crystallographic c -axis (3-fold) and making an angle of 44° with the a -axis. A translation function, and independent arguments from the Patterson series, show that this is a 2-fold axis, without translation parallel to its length. In 2 Zn insulin the 2-fold axes pass through or within 0·5 A of a 3-fold or, less probably, a 3-fold screw axis; in 4 Zn insulin they are about 1 A from it.

Mespeus—A Database of Metal Interactions with Proteins

Modeling and analogy are commonly used to identify the part that a metal may play in the structure or function of a new protein which has been recognized by structural genomics. Mespeus ( http://mespeus.bch.ed.ac.uk/MESPEUS_10/) lists metal protein interactions whose geometry has been experimentally determined and allows them to be visualized. This can contribute to the modeling process. The use of Mespeus is described with a series of examples.

Crystal structures of Leishmania mexicana phosphoglycerate mutase suggest a one-metal mechanism and a new enzyme subclass

The structures of Leishmania mexicana cofactor-independent phosphoglycerate mutase (Lm iPGAM) crystallised with the substrate 3-phosphoglycerate at high and low cobalt concentrations have been solved at 2.00- and 1.90-A resolutions. Both structures are very similar and the active site contains both 3-phosphoglycerate and 2-phosphoglycerate at equal occupancies (50%). Lm iPGAM co-crystallised with the product 2-phosphoglycerate yields the same structure. Two Co(2+) are coordinated within the active site with different geometries and affinities. The cobalt at the M1 site has a distorted octahedral geometry and is present at 100% occupancy. The M2-site Co(2+) binds with distorted tetrahedral geometry, with only partial occupancy, and coordinates with Ser75, the residue involved in phosphotransfer. When the M2 site is occupied, the side chain of Ser75 adopts a position that is unfavourable for catalysis, indicating that this site may not be occupied under physiological conditions and that catalysis may occur via a one-metal mechanism. The geometry of the M2 site suggests that it is possible for Ser75 to be activated for phosphotransfer by H-bonding to nearby residues rather than by metal coordination. The 16 active-site residues of Lm iPGAM are conserved in the Mn-dependent iPGAM from Bacillus stearothermophilus (33% overall sequence identity). However, Lm iPGAM has an inserted tyrosine (Tyr210) that causes the M2 site to diminish in size, consistent with its reduced metal affinity. Tyr210 is present in trypanosomatid and plant iPGAMs, but not in the enzymes from other organisms, indicating that there are two subclasses of iPGAMs.