Peter F. Lindley

The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centres

Abstract The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1 Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.

New perspectives on the structure and function of transferrins

Structure-function relationships for transferrins are discussed in the light of recent X-ray crystal structure determinations. A common folding pattern into two lobes, each comprising two domains is adopted; this allows the tight, but reversible binding of iron. Uptake and release of iron involve substantial domain movements which open and close the binding clefts. The iron binding sites are similar and the key role of the CO3(2-) anion bound with each Fe3+ can now be understood; structural differences near the iron binding sites suggest reasons for the different binding properties of serum transferrin and lactoferrin. The glycan moieties do not appear to affect the protein structure or metal binding properties; they are not clearly seen in the X-ray analyses but have been modelled. The accommodation of different metals and anions is illustrated by the crystal structures of Cu2+ and oxalate-substituted lactoferrins; Al3+ binding is of particular interest. New results on transferrin-receptor interactions with transferrin, and melanotransferrin and an invertebrate transferrin (both of which have defective C-terminal binding sites), emphasize possible functional differences between the two lobes. The availability of site-specific mutants of both transferrin and lactoferrin now offers the opportunity to probe the structural determinants of iron binding, iron release, and receptor binding.

An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity

Abstract The role of ceruloplasmin as a ferroxidase in the blood, mediating the release of iron from cells and its subsequent incorporation into serum transferrin, has long been the subject of speculation and debate. However, a recent X-ray crystal structure determination of human ceruloplasmin at a resolution of around 3.0 Å, in conjunction with studies associating mutations in the ceruloplasmin gene with systemic haemosiderosis in humans, has added considerable weight to the argument in favour of a ferroxidase role for this enzyme. Further X-ray studies have now been undertaken involving the binding of the cations Co(II), Fe(II), Fe(III), and Cu(II) to ceruloplasmin. These results give insights into a mechanism for ferroxidase activity in ceruloplasmin. The residues and sites involved in ferroxidation are similar to those proposed for the heavy chains of human ferritin. The nature of the ferroxidase activity of human ceruloplasmin is described in terms of its three-dimensional molecular structure.

An X-ray crystallographic study of the binding sites of the azide inhibitor and organic substrates to ceruloplasmin, a multi-copper oxidase in the plasma

Abstract Ceruloplasmin is a multi-copper oxidase, which contains most of the copper present in the plasma. It is an acute-phase reactant that exhibits a two- to three-fold increase over the normal concentration of 300 μg/ml in adult plasma. However, the precise physiological role(s) of ceruloplasmin has been the subject of intensive debate and it is likely that the enzyme has a multi-functional role, including iron oxidase activity and the oxidation of biogenic amines. The three-dimensional X-ray structure of the human enzyme was elucidated in 1996 and showed that the molecule was composed of six cupredoxin-type domains arranged in a triangular array. There are six integral copper atoms per molecule (mononuclear sites in domains 2, 4 and 6 and a trinuclear site between domains 1 and 6) and two labile sites with roughly 50% occupancy. Further structural studies on the binding of metal cations by the enzyme indicated a putative mechanism for ferroxidase activity. In this paper we report medium-resolution X-ray studies (3.0–3.5 Å) which locate the binding sites for an inhibitor (azide) and various substrates [aromatic diamines, biogenic amines and (+)-lysergic acid diethylamide, LSD]. The binding site of the azide moiety is topologically equivalent to one of the sites reported for ascorbate oxidase. However, there are two distinct binding sites for amine substrates: aromatic diamines bind on the bottom of domain 4 remote from the mononuclear copper site, whereas the biogenic amine series typified by serotonin, epinephrine and dopa bind in close vicinity to that utilised by cations in domain 6 and close to the mononuclear copper. These binding sites are discussed in terms of possible oxidative mechanisms. The binding site for LSD is also reported.

X-ray diffraction and structure of crystallins

The 3-dimensional organisation of crystallin polypeptides into globular proteins and their interactions into higher order structures are important factors governing optical functions related to refraction, accommodation and transparency. Single crystal X-ray diffraction studies have revealed the tertiary and quaternary structural organisation of β-, γ- and δ-crystallins. Regions of the lens with high refractive index contain high levels of monomeric y-crystallins while the accommodating, hydrated avian lens has largely replaced γ-crystallins with δ-crystallin. The βγ-crystallins form a superfamily of proteins of high symmetry and great diversity in which the basic building block is a 10 kD pseudo-symmetrical 2-Greek key domain. A γ-crystallin comprises two of these β-sheet domains, joined by a linker, and a short C-terminal extension. In β-crystallins the linker has an extended conformation resulting in dimer formation by a mechanism known as domain swapping. Crystallographic analysis of engineered single domains of γ-crystallins, analogous to the ancestral domain, has indicated the importance of the short C-terminal extension in directing domain pairing. γ-crystallins have numerous cysteine residues, some are conserved in the core of the protein molecule and some are variable on the protein surface. The structure of γB-crystallin has been determined at very high resolution using cryo-crystallography allowing the visualisation of the complete protein-protein and protein-water structure at the surface. β-crystallins are seen as tetramers in the crystal structures but their long sequence extensions are harder to visualise in the electron density of the hydrated crystal lattice structure. In one tight packing lattice of βB2 crystallin the N-terminal extension is seen to mediate protein-protein interactions between tetramers to form a 42 helix. The X-ray structure of the taxon-restricted avian δ-crystallin shows that the 50 kD subunit contains 22 helices that form three α-helical domains which dimerise followed by a dimer-dimer interaction to form a tetramer with a 20-helix bundle at the centre. Analysis of the spatial disposition of the sequence conserved regions showed the location of the active site cleft of the superfamily of enzymes related to δ-crystallin and argininosuccinate lyase. A different crystal structure of δ-crystallin solved under more physiological conditions revealed that tetramers assembled as higher order supramolecular helices and that the N-terminal extension may be involved. Combining the observations of higher order helical structures in both the oligomeric β-crystallin and δ-crystallin crystal lattices, we have proposed a highly speculative model for crystallin assembly in the lens fibre cells.

Iron in biology: a structural viewpoint

The element iron is essential for life and plays a number of key roles in biology. For example, iron is the most abundant metal in humans with healthy adults possessing some 3 to 4 g. The bulk of this iron is bound to the oxygen transporting haemoglobin in the red blood corpuscles, to the muscle oxygen storage protein myoglobin, or is stored by ferritin and haemosiderin. Less than 1% of this iron is bound to the various iron enzymes and redox proteins or is being transported through the blood by transferrin. In Nature, many redox reactions are dependent on iron-containing enzymes whereby electron transport is facilitated by changes in the oxidation state of the metal. Nitrogen fixation and photosynthesis are examples of processes in which iron-containing enzymes play vital roles. This review describes the three-dimensional molecular structures of some of the more important of these iron proteins and enzymes and attempts to explain their functional roles in terms of these structures. Much of the structural information has been obtained using macromolecular x-ray crystallographic methods. The three-dimensional molecular structures provide important templates which can be used to incorporate structural information obtained by complementary techniques (biochemical, molecular biological, biophysical and spectroscopic), to form a comprehensive model for interpreting structure - function relationships. The first part of the review briefly describes recent developments in macromolecular crystallography, including the vital role played by synchrotron x-radiation sources.

The Haemophilia A Mutation Search Test and Resource Site, Home Page of the Factor VIII Mutation Database: HAMSTeRS

In order to facilitate easy access to and aid understanding of the causes of haemophilia A at the molecular level we have constructed HAMSTeRS, the third release of the factor VIII mutation database and the first release of this database that may be accessed and interrogated over the internet through a World Wide Web browser. The database also presents a review of the structure and function of factor VIII and the molecular genetics of haemophilia A, a real time update of the biostatistics of each parameter in the database, a molecular model of the A1, A2 and A3 domains of the factor VIII protein (based on the crystal structure of caeruloplasmin) and a bulletin board for discussion of issues in the molecular biology of factor VIII.

The use of synchrotron radiation in protein crystallography

Synchrotron radiation provides a source of high intensity, wavelength tuneable, highly collimated radiation which can be used to investigate structure-function relationships in biological macromolecules in a number of ways. This article will deal mainly with the applications of the hard X-ray region (0.5 – 2.5 A) of the synchrotron spectrum to three-dimensional structure analysis of single crystals of macromolecules. The main themes highlighted will be, (a) why synchrotron radiation is necessary for structural studies on biological macromolecules, (b) examples of experimental stations used for protein crystallography at the Synchrotron Radiation Source, DRAL Daresbury Laboratory, (c) how the wavelength tunability can be exploited to solve the “phase problem”, (d) high resolution data using cryogenic methods and (e) studies on large structures.

The Search for A “Prismane” Fe–S Protein

Publisher Summary Iron–sulfur (Fe–S) clusters are found throughout nature. They usually function in electron transfer reactions and are found in small molecules, such as the ferredoxins and in redox enzymes, where they shuttle electrons to or from the active site. They contain iron and inorganic sulfur atoms and are bound to the protein, through the Fe atoms, primarily by the sulfur atoms of cysteine residues, but occasionally by histidine nitrogen or by aspartate oxygen atoms. The simplest Fe–S cluster contains one Fe atom ligated by four cysteine sulfurs, although this is not a true Fe–S cluster, because it lacks inorganic sulfur. This chapter presents an overview of the discovery of the Fepr protein, the spectroscopy that led to the suggestion that it contained a [6Fe–6S] cluster, and the subsequent crystal structure analysis that disproved this hypothesis, yet uncovered what is at a present a unique Fe–S cluster in biology.